Gastrointestinal proliferative factor and uses thereof

ABSTRACT

The invention relates to pharmaceutical compositions comprising gastrointestinal proliferative factor (GIPF) polynucleotides and polypeptides. The invention further relates to the therapeutic use of GIPF to prevent or treat conditions or disorders associated with the degeneration of the epithelial mucosa.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/046,644, filed Jan. 27, 2005 from which priority is claimedpursuant to 35 U.S.C. §120, and claims benefit under 35 U.S.C. §119(e)of U.S. Provisional Application No. 60/539,605, filed Jan. 27, 2004 andU.S. Provisional Application No. 60/619,241, filed Oct. 15, 2004, whichapplications are hereby incorporated by reference in their entireties.

1. BACKGROUND

1.1 Field of the Invention

The present invention relates generally to compositions that comprisegastrointestinal proliferation factor polypeptides and polynucleotides,and methods for using the same.

1.2 Sequence Listing

A sequence listing is provided.

1.3 Background

Ionizing radiation therapy and cytotoxic chemotherapy produce injuriesto the oral and gastrointestinal mucosa, which remain significantproblems for patients undergoing antineoplastic treatments. Mucositis isthe inflammation of the mucous membranes and is a particularly commonproblem in this patient population due to the use of chemotherapy andradiation therapy used with curative or palliative intent. The mucosalinjuries to the gastrointestinal tract seen with radiation andchemotherapy (to these areas) include the destruction of crypt cells, adecrease in villous height and ulceration and necrosis of thegastrointestinal epithelium (Berthrong M, World J Surg 10:155-170(1986)), which underlie disorders including gastrointestinal mucositisand enterocolitis. To the patients this can mean abdominal pain, bloodydiarrhea, malabsorption and in some cases bacterial translocation(Guzman et al., J Surg Res 46:104-107 (1989)). In addition, chemotherapyand ionizing radiation can affect other mucous membranes including thoseof the oropharynx and lips, and those of the esophagus. It is well knownthat combined modality therapy of concurrent radiation and chemotherapycan produce highly symptomatic stomatitis in patients with head and neckcancer, and esophagitis in patients with small cell lung cancer.

Chemotherapy and radiation therapy cause injury to the oral andgastrointestinal mucosa through direct and indirect toxicity. Themechanism for direct mucositis is nonspecific cell killing of rapidlydividing basal epithelial cells that results in epithelial thinning,inflammation, decreased cell renewal, and ultimately ulceration. Thesepainful lesions also produce an increased risk for local and systemicinfection. Indirect mucotoxicity is a byproduct of chemotherapy-inducedmyelosuppression, which permits bacterial and viral infections at thesite of direct mucosal injury. The severity of these effects maypreclude dose escalation, delay treatment, and warrant dose reductions,thus limiting the effectiveness of cancer therapy.

Prophylaxis and therapy for chemotherapy and radiation therapy-induced(mucosal) gastrointestinal injuries (mucositis) commonly entailsprescription of suboptimal doses of chemotherapy or radiotherapy, adownward dose modification in subsequent treatment courses followingtoxicity, or the use of specific antidotes such as leucovorin aftermoderate-dose or high-dose methotrexate (Allegra CJ. Antifolates. In:Chabner and Collins, eds. Cancer Chemotherapy: Principles and Practice.Philadelphia, Pa. JP Lippincoft Co; 1990:110-153.)

Injury to the gastrointestinal mucosa is also associated with chronicinflammatory disorders of the gastrointestinal tract, which arecollectively referred to as inflammatory bowel disease. Cytokine-basedtherapies are available for the treatment of inflammatory bowel disease(Bouma and Strober Nature Rev 3:521-533 (2003)). However, resection ofthe small intestine is often indicated in patients with inflammatorybowel disease such as Crohn's disease. Surgical resection of the smallintestine may also be necessary following traumatic injury, vascularaccidents, and cancer. Surgical resection that leaves less than 200 cmof viable small bowel places a patient at risk for developingshort-bowel syndrome (SBS). SBS is a disorder that is clinically definedby malabsorption, diarrhea, fluid and electrolyte disturbances, andmalnutrition. The management of patients with SBS frequently requireslong-term, if not life long use of parenteral nutrition (DiBaise et al.,Am J Gastroenterol 99:1823-1832 (2004)).

Thus, there is a need to find agents that may be used prophylacticallyor therapeutically to increase the tolerance to antineoplastictreatments, to advance current therapies for treating inflammatory boweldisease, and to restore the digestive and absorptive processes that arecompromised following surgical resection of the intestine.

2. SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery thatGastroIntestinal Proliferative Factor (GIPF) induces the proliferationof epithelial cells of the gastrointestinal tract. Thus, compositionscomprising GIPF, fragments or analogs thereof, may be used for thetreatment of conditions where epithelialization is desirable, such asfor the treatment of gastrointestinal disorders including chemotherapyand radiation therapy-induced mucositis, mucositis of the oropharynx,lips and esophagus, inflammatory bowel disease, and other conditionsincluding wounds, burns, ophthalmic disorders, and any disorder wherestimulation of epithelial cell proliferation or regeneration is desired.

Accordingly, in one embodiment, the invention is directed to acomposition comprising a therapeutically effective amount of a GIPFpolypeptide and a pharmaceutically acceptable carrier.

The compositions of the present invention include isolatedpolynucleotides encoding GIPF polypeptides, including recombinant DNAmolecules, and cloned genes or degenerate variants thereof, especiallynaturally occurring variants such as allelic variants. Specifically, thepolynucleotides of the present invention are based on a GIPFpolynucleotide isolated from a cDNA library prepared from human fetalskin mRNA (SEQ ID NO: 2).

The compositions of the present invention also include vectors such asexpression vectors containing the polynucleotides of the invention,cells genetically engineered to contain such polynucleotides and cellsgenetically engineered to express such polynucleotides.

The compositions of the invention comprise isolated polynucleotides thatinclude, but are not limited to, a GIPF polynucleotide, a fragment, orvariant thereof; a polynucleotide comprising the full length proteincoding sequence of SEQ ID NO: 2 or 3 (for example, SEQ ID NO: 4;GIPFwt); a polynucleotide comprising the V5-His-tagged protein codingsequence of SEQ ID NO: 5 (for example SEQ ID NO: 6; GIPFt); apolynucleotide comprising the nucleotide sequence of the dominant matureprotein coding sequence of SEQ ID NO: 9 (for example SEQ ID NO: 10); apolynucleotide comprising the nucleotide sequence of the mature proteincoding sequence of SEQ ID NO: 11 (for example SEQ ID NO: 12); apolynucleotide comprising the nucleotide sequence of the thrombospondindomain of SEQ ID NO: 13 (for example SEQ ID NO: 14); a polynucleotide ofSEQ ID NO: 15 comprising the nucleotide sequence that encodes a dominantmature protein sequence that lacks the furin cleavage site (for exampleSEQ ID NO: 16); the polynucleotide of SEQ ID NO: 17 comprising thenucleotide sequence that encodes a GIPF polypeptide that comprises amutated furin cleavage site (SEQ ID NO: 18); and polynucleotides thatencode GIPF polypeptides that comprise varying lengths of thefull-length GIPF (SEQ ID NOs: 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104 and 177). The polynucleotide compositions of the present inventionalso include, but are not limited to, a polynucleotide that hybridizesunder stringent hybridization conditions to (a) the complement of any ofthe nucleotide sequences set forth in SEQ ID NO: 2, 3, 5, 9, 11, 13, 15,17, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104 or 177; (b) anucleotide sequence encoding any of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16,18, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or 178; apolynucleotide which is an variant (e.g., an allelic variant) of anypolynucleotides recited above having at least 70% (e.g., 75%, 80%, 85%,90%, 92%, 94%, 96%, 98%, or 99%) polynucleotide sequence identity to thepolynucleotides; a polynucleotide which encodes a species homolog (e.g.,an ortholog) of any of the polypeptides recited above; or apolynucleotide that encodes a polypeptide comprising a specific domainor truncation of the polypeptide of SEQ ID NO: 4 or 6.

This invention further provides cloning or expression vectors comprisingat least a fragment of the polynucleotides set forth above and hostcells or organisms transformed with these expression vectors. Usefulvectors include plasmids, cosmids, lambda phage derivatives, phagemids,and the like, that are well known in the art. Accordingly, the inventionalso provides a vector including a polynucleotide of the invention and ahost cell containing the polynucleotide. In general, the vector containsan origin of replication functional in at least one organism, convenientrestriction endonuclease sites, and a selectable marker for the hostcell. Vectors according to the invention include expression vectors,replication vectors, probe generation vectors, and sequencing vectors. Ahost cell according to the invention can be a prokaryotic or eukaryoticcell and can be a unicellular organism or part of a multicellularorganism.

The pharmaceutical compositions of the present invention includepolypeptides comprising, but not limited to, an isolated polypeptideselected from the group comprising the amino acid sequence of SEQ ID NO:4, 6, 10, 12, 14, 16, 18, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105or 178. Polypeptides of the invention also include polypeptides withbiological activity that are encoded by (a) any of the polynucleotideshaving a nucleotide sequence set forth in the SEQ ID NO: 2, 3, 5, 9, 11,13, 15, 17, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 177 above; or(b) polynucleotides that hybridize to the complement of thepolynucleotides of (a) under stringent hybridization conditions.Biologically or immunologically active analogs of any of the proteinsequences listed as SEQ ID NO: 4, 6, 10, 12, 14, 16, 18, 85, 87, 89, 91,93, 95, 97, 99, 101, 103, 105 or 178, and substantial equivalentsthereof that retain biological are also contemplated. The polypeptidesof the invention may be wholly or partially chemically synthesized butare preferably produced by recombinant means using the geneticallyengineered cells (e.g. host cells) of the invention. The inventionincludes polypeptides that are at least 85%, 90%, 92%, 94%, 96%, 98%, or99% identical to any of SEQ ID NO: 4, 6, 10, 12, 14, 16, 18, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105 and 178. The invention also includespolypeptides that differ in sequence from any of SEQ ID NO: 4, 6, 10,12, 14, 16, 18, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 178and at 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues. Theamino acid changes can be conservative or non-conservative.

The invention also relates to methods for producing a GIPF polypeptidecomprising culturing host cells comprising an expression vectorcontaining at least a fragment of a GIPF polynucleotide encoding theGIPF polypeptide of the invention in a suitable culture medium underconditions permitting expression of the desired polypeptide, andpurifying the protein or peptide from the culture or from the hostcells. Preferred embodiments include those in which the protein producedby such a process is a mature or dominant mature form of the protein.

The polypeptides according to the invention can be used in a variety ofconventional procedures and methods that are currently applied to otherproteins. For example, a polypeptide of the invention can be used togenerate an antibody that specifically binds the polypeptide. Suchantibodies, particularly monoclonal antibodies, are useful for detectingor quantifying the polypeptide in tissue.

In further embodiments, the subject invention is directed to a method ofstimulating epithelial cell proliferation. The method comprisescontacting epithelial cells with a composition that includes atherapeutically effective amount of a GIPF polypeptide, fragment oranalog thereof, and a pharmaceutically acceptable carrier. Specifically,a subject in need of stimulation (including cytoprotection,proliferation and/or differentiation) of epithelial cells will beadministered therapeutically-effective or prophylactically-effectiveamounts of GIPF protein, fragments or analogs thereof.

In all the methods described, epithelial cells may be contacted with theGIPF polypeptides in vitro or in vivo.

Methods are also provided for preventing, treating, or ameliorating amedical condition which comprises the step of administering to amammalian subject a therapeutically effective amount of a compositioncomprising a peptide of the present invention and a pharmaceuticallyacceptable carrier.

In particular, the GIPF polypeptides of the invention may be used toinduce the proliferation and/or differentiation of gastrointestinalcrypt cells to regenerate the epithelial layer of the alimentary tract.Thus, the GIPF polypeptides and polynucleotides of the invention may beused in the treatment of chemotherapy or radiation therapy-inducedmucositis and enterocolitis, and inflammatory bowel disease. They mayalso be used in the treatment of diseases, and other conditionsincluding wounds, burns, ophthalmic disorders, and any disorder wherestimulation of epithelial cell proliferation or regeneration is desired.

Polynucleotides and polypeptides of the invention may also be used asmarkers of differentiation and development of gastrointestinalepithelium.

The methods of the invention also provide methods for the treatment ofdisorders as recited herein which comprise the administration of atherapeutically effective amount of a composition comprising apolynucleotide or polypeptide of the invention and a pharmaceuticallyacceptable carrier to a mammalian subject exhibiting symptoms ortendencies related to disorders as recited herein. In addition, theinvention encompasses methods for treating diseases or disorders asrecited herein comprising the step of administering a compositioncomprising compounds and other substances that modulate the overallactivity of the target gene products and a pharmaceutically acceptablecarrier. Compounds and other substances can effect such modulationeither on the level of target gene/protein expression or target proteinactivity. Specifically, methods are provided for preventing, treating orameliorating a medical condition, including mucositis and inflammatorybowel disease, wounds, which comprises administering to a mammaliansubject, including but not limited to humans, a therapeuticallyeffective amount of a composition comprising a polypeptide of theinvention or a therapeutically effective amount of a compositioncomprising a binding partner of GIPF polypeptides of the invention. Themechanics of the particular condition or pathology will dictate whetherthe polypeptides of the invention or binding partners of these would bebeneficial to the individual in need of treatment.

The invention further provides methods for manufacturing medicamentsuseful in the above-described methods.

The present invention further relates to methods for detecting thepresence of the polynucleotides or polypeptides of the invention in asample (e.g., tissue or sample). Such methods can, for example, beutilized as part of prognostic and diagnostic evaluation of disorders asrecited herein and for the identification of subjects exhibiting apredisposition to such conditions.

The invention provides a method for detecting a polypeptide of theinvention in a sample comprising contacting the sample with a compoundthat binds to and forms a complex with the polypeptide under conditionsand for a period sufficient to form the complex and detecting formationof the complex, so that if a complex is formed, the polypeptide isdetected.

The invention also provides kits comprising polynucleotide probes and/ormonoclonal antibodies, and optionally quantitative standards, forcarrying out methods of the invention. Furthermore, the inventionprovides methods for evaluating the efficacy of drugs, and monitoringthe progress of patients, involved in clinical trials for the treatmentof disorders as recited above.

The invention also provides methods for the identification of compoundsthat modulate (i.e., increase or decrease) the expression or activity ofthe polynucleotides and/or polypeptides of the invention. Such methodscan be utilized, for example, for the identification of compounds thatcan enhance the therapeutic activity of the GIPF polypeptides, andameliorate symptoms of disorders as recited herein. Such methods caninclude, but are not limited to, assays for identifying compounds andother substances that interact with (e.g., bind to) the polypeptides ofthe invention.

The invention provides a method for identifying a compound that binds tothe polypeptide of the present invention comprising contacting thecompound with the polypeptide under conditions and for a time sufficientto form a polypeptide/compound complex and detecting the complex, sothat if the polypeptide/compound complex is detected, a compound thatbinds to the polypeptide is identified.

Also provided is a method for identifying a compound that binds to thepolypeptide comprising contacting the compound with the polypeptide in acell for a time sufficient to form a polypeptide/compound complexwherein the complex drives expression of a reporter gene sequence in thecell and detecting the complex by detecting reporter gene sequenceexpression so that if the polypeptide/compound complex is detected acompound that binds to the polypeptide is identified.

Another embodiment of the invention provides gene therapy by delivery ofGIPF polypeptides for the treatment of conditions or disorders recitedherein.

In a related embodiment, the invention is directed to use of a vectorcomprising a gene encoding a GIPF polypeptide operably associated withan expression control sequence that provides for expression of the GIPFpolypeptide in the manufacture of a medicament for treating disorders asrecited herein. More particularly, the invention provides for use of anadenoviral vector of the invention, e.g., as set out below, in themanufacture of a medicament for treating mucositis or inflammatory boweldisease.

In addition to the foregoing methods and uses, the invention provides anovel virus vector comprising a gene encoding a GIPF polypeptideoperably associated with an expression control sequence. In a preferredembodiment, the virus vector is an adenovirus vector. The virus vectorsof the invention can provide a gene encoding any GIPF polypeptide, asset forth above.

The invention further provides a pharmaceutical composition comprisingany of the virus vectors of the invention and a pharmaceuticallyacceptable carrier.

In yet another aspect, the invention concerns a transgene constructcomprising a nucleic acid encoding a native human GIPF protein, analogor a fragment thereof, under the control of transcriptional regulatorysequences directing its expression to B-cells. The transgene constructpreferably comprises a B-cell specific promoter, such as animmunoglobulin kappa chain promoter.

In another aspect, the invention concerns a transgenic non-human mammalthat produces in its B-cells detectable levels of a native human GIPFprotein, analog or a fragment thereof, wherein said transgenic mammalhas stably integrated into its genome a nucleic acid sequence encoding anative human GIPF protein, analog or a fragment thereof having thebiological activity of native human GIPF, operably linked totranscriptional regulatory sequences directing its expression toB-cells. The transcriptional regulatory sequences preferably comprise aB-cell specific promoter, such as the immunoglobulin kappa chainpromoter. Without limitation, the non-human transgenic mammal may, forexample, be mouse, rat, rabbit, pig, sheep, goat or cattle.

In another aspect the invention concerns a method of screening drugcandidates for the treatment of a disease or disorder recited hereincomprising (a) administering a drug candidate to a transgenic mouse thatexpresses in its B-cells a GIPF polypeptide, and develops intestinaldistension associated with hyperproliferation of epithelial cells, and(b) evaluating the effect of the candidate drug on thehyperproliferation of the epithelial cells. The drug candidates maymodulate (i.e. increase or decrease) the expression or activity of thepolynucleotides and/or polypeptides of the invention.

The invention also includes a method of treating or ameliorating amedical condition, including mucositis and inflammatory bowel disease,and wounds, which comprises administering to a mammalian subject,including but not limited to humans, a therapeutically effective amountof a GIPF polypeptide together with a cytokine.

In another aspect the invention includes pharmaceutical compositionscomprising a polypeptide of the invention, a second therapeutic agent,e.g., a cytokine, and a pharmaceutically acceptable carrier.

The invention features, a composition comprising a therapeuticallyeffective amount of a GIPF polypeptide, fragment, or analog thereof, anda pharmaceutically acceptable carrier.

The invention also features a pharmaceutical composition comprising apolypeptide comprising a biologically active fragment of GIPF and apharmaceutically acceptable carrier. In various embodiments, the GIPF ishuman GIPF; and the polypeptide comprises a biologically active fragmentof the polypeptide of SEQ ID NO:4.

In another embodiment the invention features a pharmaceuticalcomposition comprising a polypeptide comprising a polypeptide fragmentof SEQ ID NO:4 wherein the polypeptide fragment comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 10, 12, 14,16, 18, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, and 178.

In various embodiments of the compositions and methods, the polypeptideis glycosylated; the polypeptide is not glycosylated; the polypeptidestimulates epithelial cell proliferation; and the polypeptide comprisesan amino acid sequence that is at least 80% identical to the amino acidsequence of SEQ ID NO:4.

The invention also features: a method of stimulating epithelial cellproliferation in a subject comprising administering to said subject acomposition comprising a GIPF polypeptide, fragment or analog thereofand a carrier; a method of treatment comprising administering to amammalian subject in need thereof a therapeutically effective amount ofa composition comprising a GIPF polypeptide and a pharmaceuticallyacceptable carrier; a method of treating mucositis, inflammatory boweldisease, or short bowel syndrome comprising administering to a mammaliansubject in need thereof a therapeutically effective amount of acomposition comprising a GIPF polypeptide and a pharmaceuticallyacceptable carrier; a method for stimulating epithelial cellproliferation in the gastrointestinal tract of a patient, the methodcomprising administering a therapeutically effective amount of acomposition comprising a GIPF polypeptide and a pharmaceuticallyacceptable carrier. In various embodiments: epithelial cellproliferation in the esophagus is stimulated, epithelial cellproliferation in the small intestine is stimulated; epithelial cellproliferation in the large intestine is stimulated; epithelial cellproliferation in the oral cavity is stimulated and epithelial cellproliferation in the stomach is stimulated.

The invention also features a method for treating a patient at risk fordamage to epithelial cells lining at least a portion of thegastrointestinal tract, the method comprising administering atherapeutically effective amount of a composition comprising a GIPFpolypeptide and a pharmaceutically acceptable carrier. In certainembodiments: the patient has undergone or will undergo radiation therapyand the patient has undergone or will undergo chemotherapy.

The invention includes a method for treating a patient that hasundergone radiation therapy or chemotherapy comprising administering atherapeutically effective amount of a composition comprising a GIPFpolypeptide and a pharmaceutically acceptable carrier.

In other embodiments, the invention features an adenoviral vectorcomprising a gene encoding GIPF operably associated with an expressioncontrol sequence as well as a pharmaceutical composition comprising sucha vector.

In other embodiments, the invention features a method of stimulatingepithelial cell proliferation in a subject comprising administering tosaid subject the pharmaceutical composition comprising features anadenoviral vector comprising a gene encoding GIPF operably associatedwith an expression control sequence.

The invention also features a transgene construct comprising a nucleicacid encoding a GIPF protein, wherein said nucleic acid is operablylinked to transcriptional regulatory sequences directing its expressionin B-cells. In certain embodiments, the transgene construct comprises aB-cell specific promoter.

The invention also features a transgenic mouse that produces in itsB-cells cells detectable levels of a native human GIPF protein, whereinsaid transgenic mouse has stably integrated into its genome a nucleicacid sequence encoding a GIPF protein, operably linked totranscriptional regulatory sequences directing its expression toB-cells. In some embodiments, the transcriptional regulatory sequencescomprise a B-cell promoter.

The invention features a method of identifying a drug candidate for thetreatment of mucositis, inflammatory bowel disease or short bowelsyndrome, comprising:

(a) administering a test compound to a transgenic mouse that expressesin its B-cells a recombinant GIPF polypeptide and exhibits increasedintestinal epithelial cell proliferation compared to an otherwiseidentical mouse not expressing a recombinant GIPF polypeptide intestinalepithelial cell proliferation; and

(b) evaluating the effect of said test compound on intestinal epithelialcell proliferation, wherein an increase in intestinal cell proliferationidentifies the test compound as a drug candidate for the treatment ofmucositis, inflammatory bowel disease or short bowel syndrome. Incertain embodiments, the intestinal epithelial cell is a crypt cell.

The invention also features: an isolated polynucleotide selected fromthe group consisting of SEQ ID NO: 9, 11, 13, 15, 17, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, and 104; an isolated polynucleotide encoding apolypeptide with biological activity, said polynucleotide having greaterthan about 95% sequence identity to polynucleotide selected from thegroup consisting of SEQ ID NO: 9, 11, 13, 15, 17, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, and 104.

The invention also features: an isolated polypeptide selected from thegroup consisting of SEQ ID NO: 12, 14, 16, 18, 85, 87, 89, 91, 93, 95,97, 99, 101, 103, and 105; and an isolated polypeptide comprising anamino acid sequence which is at least 95% identical to the amino acidsequence selected from the group consisting of SEQ ID NO: 12, 14, 16,18, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, and 105.

The invention further features an expression vector comprisingexpression regulatory elements operatively linked to a polynucleotide ofSEQ ID NO: 5, 9, 11, 13, 15, 17, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, and 104.

The invention also features a host cell transformed or transfected witha polynucleotide of SEQ ID NO: 5, 9, 11, 13, 15, 17, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104 or 177. In some embodiments the cell isprokaryotic, in others it is eukaryotic.

The invention also features a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO: 12, 14, 16, 18, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, or 105, the method comprising:

(a) culturing an isolated cell comprising a nucleic acid moleculeencoding a polypeptide of SEQ ID NO: 12, 14, 16, 18, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, or 105 in culture medium under conditions suitablefor expressing the polypeptide; and

(b) purifying the polypeptide from the cell or the culture medium.

The invention features a method for producing a pharmaceuticalcomposition comprising a polypeptide comprising the amino acid sequenceof SEQ ID NO: 12, 14, 16, 18, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,105 or 178, the method comprising

(a) culturing an isolated cell comprising a nucleic acid moleculeencoding a polypeptide of SEQ ID NO: 12, 14, 16, 18, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, or 178 in culture medium under conditionssuitable for expressing the polypeptide;

(b) purifying the polypeptide from the cell or the culture medium; and

(c) combining the purified polypeptide with a pharmaceuticallyacceptable carrier.

The invention features a method for producing a pharmaceuticalcomposition comprising a polypeptide comprising the amino acid sequenceof SEQ ID NO: 12, 14, 16, 18, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,105 or 178, the method comprising

(a) synthesizing comprising a polypeptide comprising the amino acidsequence of SEQ ID NO: 12, 14, 16, 18, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105 or 178;

(b) purifying the polypeptide; and

(c) combining the purified polypeptide with a pharmaceuticallyacceptable carrier.

The invention also features an expression vector construct comprising anucleic acid encoding a GIPF protein, wherein the nucleic acid isoperably linked to transcriptional regulatory sequences directingexpression of the GIPF protein in intestinal epithelial cells.

In another aspect the invention includes a transgenic mouse thatproduces in its intestinal epithelial cells detectable levels of a GIPFprotein, wherein said transgenic mouse has stably integrated into itsgenome a nucleic acid sequence encoding a GIPF protein, wherein thenucleic acid sequence encoding the GIPF protein is operably linked totranscriptional regulatory sequences directing expression of the GIPFprotein in intestinal epithelial cells.

In another aspect the invention features an expression vector constructcomprising a nucleic acid encoding a GIPF protein and a Wnt3a protein,wherein the nucleic acid is operably linked to transcriptionalregulatory sequences directing its expression in intestinal epithelialcells.

The invention also features a transgenic mouse that produces in itsintestinal epithelial cells detectable levels of a native GIPF and Wnt3aprotein, wherein said transgenic mouse has stably integrated into itsgenome a nucleic acid sequence encoding a GIPF protein, operably linkedto transcriptional regulatory sequences directing expression of the GIPFprotein in intestinal epithelial cells.

In some embodiments the transgenic mouse exhibits intestinal distension.

Additional aspects and advantages of the invention will be apparent tothose skilled in the art upon consideration of the followingdescription, which details the practice of the invention.

3. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the DNA sequence (SEQ ID NO: 2) (A) and correspondingamino acid sequence for the full-length GIPF (SEQ ID NO: 4) (B). SEQ IDNO: 2 includes the 5 prime and 3 prime untranslated regions inconjunction with the open reading frame.

FIG. 2 depicts the expression of GIPF mRNA in tissues from human (A) andmouse (B).

FIG. 3 is a schematic representation of the GIPF polypeptides of thecompositions of the invention. The numbers that are underlinedcorrespond to the SEQ ID NOs of the polypeptides, and the remainingnumbers are the SEQ ID NOs of the encoding polynucleotide sequences.

FIG. 4A shows the BLASTP amino acid sequence alignment between the GIPFprotein encoded by SEQ ID NO: 2 or 3 (i.e. SEQ ID NO: 4) and human stemcell growth factor A1 SEQ ID NO: 23 (SEQ ID NO: 10 from PCT WO 01/77169A2), indicates that the two sequences share 63% similarity over aminoacid residues 10 through 251 of SEQ ID NO: 4 and amino acid residues 11through 257 of SEQ ID NO: 23, and 46% identity over the amino acidresidues 10 through 251 of SEQ ID NO: 4 and amino acid residues 11through 257 of SEQ ID NO: 23.

FIG. 4B shows the BLASTP amino acid sequence alignment between the GIPFprotein encoded by SEQ ID NO: 2 or 3 (i.e. SEQ ID NO: 4) GIPFpolypeptide and a specific region of human thrombospondin 1 (amino acidresidues 501 through 657 of SwissProt accession number P07996; SEQ IDNO: 28). The figure indicates that the two sequences share 36%similarity and 26% identity over amino acid residues 14 through 166 ofSEQ ID NO: 4 and amino acid residues 501 through 657 of SEQ ID NO: 28,wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are presented as dashes.

FIGS. 5A-5R depicts the steps of the method used to generate theGIPF-knock-in (GIPF-KI) vector of the invention. A preferred method forgenerating transgenic mice that express GIPF in their B cells is alsodescribed.

FIG. 6 depicts the location of the probe utilized in Southern blotanalysis to select ES clones resulting from homologous recombination, aswell as the EcoRI digest fragment sizes of mouse genomic DNA that hasundergone homologous or non-homologous recombination.

FIG. 7 shows the gross pathology of the intestinal tract of the GIPF-KImice: control (A) GIPF-KI (B).

FIG. 8 H&E staining of transverse sections of small intestine of GIPF-KI(A) and control chimeric (B) mice, respectively.

FIG. 9 shows H&E staining of the intestinal sections of FIG. 8 seenunder higher magnification. Panels A and C correspond to the GIPF-KIsection seen in panel A of FIG. 8, and panels B and D correspond to theintestinal section derived from a control chimeric mouse seen in panel Bof FIG. 8.

FIG. 10 shows Ki67 staining of cross-sections of the small intestinefrom a control chimeric mouse (A and C), and from a GIPF-KI mouse (B andD).

FIG. 11 Cross-sections of small intestine derived from a control mouse(A and C), and from a mouse treated with 1×10¹⁰ viral particles (VP).The sections were obtained three days following injection of the emptyor GIPF adenovirus, respectively.

FIG. 12 Cross-sections of small intestine derived from control (A andC), and from a mouse treated with 1×10¹⁰ viral particles (VP) (FIGS. 12B and D). The sections were obtained five days following injection ofthe empty or GIPF adenovirus, respectively.

FIG. 13 Incorporation of BrdU into proliferating crypt cells of thesmall intestine of control mice (A and C) and mice treated with 1×10¹⁰viral particles (VP) (B and D).

FIG. 14 Ki67 staining of proliferating crypt cells of the smallintestine of control (A and C) and GIPF-adenovirus-treated mice (B andD).

FIG. 15 H&E staining of cross sections derived from the small intestineof control mice (A and C) and mice that had been treated withGIPF-adenovirus at 1×10⁹ viral particles (B and D).

FIG. 16 H&E staining of cross-sections derived from the colon of control(A and C) and GIPF-adenovirus-treated mice (B and D).

FIG. 17 Solubility requirements of native V5-His-tagged GIPF proteinpurified from CHO cells.

FIG. 18 Pharmacokinetics of V5-His-tagged GIPF protein in mouse serum.

FIG. 19 H&E staining of cross sections derived from the small intestineof control mice (A and C) and mice that had been treated with purifiedGIPF protein (B and D).

FIG. 20 Incorporation of BrdU into proliferating crypt cells of thesmall intestine of control mice (A and C) and mice that had been treatedwith purified GIPF protein (B and D).

FIG. 21 H&E staining of cross sections derived from the colon of controlmice (A) and mice that had been treated with purified GIPF protein (B).

FIG. 22 Incorporation of BrdU into proliferating crypt cells of thecolon of control mice (A and C) and mice that had been treated withpurified GIPF protein (B and D).

FIG. 23 H&E staining of cross sections derived from the small intestineof non-irradiated mice (A), irradiated mice treated with saline (B), KGF(C) or GIPFwt (D).

FIG. 24 Effect of 5-FU on the size of tumors in control mice and micereceiving GIPFwt.

FIG. 25 Effect of GIPF on the gross pathology of the small intestine andcolon of normal (E and F) and tumor-bearing mice (A-D).

FIG. 26 H&E staining of cross sections derived from the small intestineand colon of normal and tumor-bearing mice that had received 5-FU and/orGIPF.

FIG. 27 Micromorphometry measurements of the villus height and cryptdepth show the effect of GIPF on the intestinal epithelium of mice thatreceived 5-FU.

FIG. 28 Ki67 staining of proliferating epithelial cells of the ventraltongue of control mice (A and B), and mice treated with KGF or GIPF(C-E) and submitted to total body irradiation.

FIG. 29 Ki67 staining of proliferating epithelial cells of the dorsaltongue of control mice (A and B), and mice treated with KGF or GIPF (Cand D) and submitted to total body irradiation.

FIG. 30 Proliferative index of ventral tongue epithelium from micetreated with KGF or GIPF and submitted to total body irradiation.

FIG. 31 H&E staining of sections derived from the tongue of mice treatedwith GIPF and submitted to total body irradiation.

FIG. 32 Effect of GIPF on the inflammatory bowel disease activity index(IBDAI) of mice with DSS-iduced colitis.

FIG. 33 Effect of GIPF on the score for animal body weight in mice withDSS-induced colitis.

FIG. 34 Effect of GIPF on the score for stool consistency in mice withDSS-induced colitis.

FIG. 35 Effect of GIPF on the score for rectal bleeding in mice withDSS-induced colitis.

FIG. 36 Effect of GIPF on the gross pathology of the small intestine andcolon of control and DSS-treated mice.

FIG. 37 H&E staining of cross sections derived from the small intestineand colon of mice that had received DSS and/or GIPF.

FIG. 38 Micromorphometry measurements of the villus height and cryptdepth show the effect of GIPF on the intestinal epithelium of mice withDSS-induced colitis.

FIG. 39 Incorporation of BrdU into proliferating crypt cells of thesmall intestine and colon of mice that had received DSS and/or GIPF.

FIG. 40 Effect of GIPF on the proliferation of the small intestinalepithelium of mice with DSS-induced colitis.

FIG. 41 Effect of GIPF on the stabilization of β-catenin in humanendocrinic and kidney epithelial cells. GIPF induced the dose-dependent(A) and time-dependent (B) stabilization of β-catenin in HEK293 cells.The stabilizing effect of GIPF is not disrupted by boiling (C).

FIG. 42 Effect of GIPF on the phosphorylation of GSK3β.

FIG. 43 Schematic representation GIPF polypeptide analogs designed todetermine the ability of various regions of GIPF in stabilizingβ-catenin. The fragment numbers 1-11 respectively correspond topolypeptide SEQ ID NOs; 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, and105.

FIG. 44 Stabilization of β-catenin by the GIPF analogs depicted in FIG.43.

FIG. 45 Comparison of the activity of human and mouse GIPF on the grosspathology of mouse intestines.

FIG. 46 Effect of GIPF on intestinal crypt depth.

FIG. 47 Effect of GIPF on the stabilization of β-catenin in isolatedcrypt cells.

FIG. 48 Effect of GIPF on body weight of animals with TNBS-inducedcolitis.

FIG. 49 Effect of GIPF on the colitis score in animals with TNBS-inducedcolitis.

FIG. 50 Effect of GIPF on chronic colitis induced by DSS.

FIG. 51 Effect of GIPF on villus height and crypt depth in animals withDSS-induced chronic colitis.

FIG. 52 Effect of GIPF on the crypt proliferative index of animals withDSS-induced chronic colitis.

FIG. 53 Effect of GIPF on the survival of crypts following irradiation.

FIG. 54 A-N Diagrammatic representation of the construction of atransgene for the villin-driven expression of GIPF in the epithelium oftransgenic mice.

FIG. 55 Embryonic expression of GIPF in the intestinal epithelium andliver of transgenic mice.

FIG. 56 Stabilization of β-catenin in transgenic mice that express GIPF.

FIG. 57 H&E staining of sections of the small intestine of transgenicmice that express GIPF.

FIG. 58 A-C Diagrammatic representation of the construction of atransgene for the villin-driven expression of GIPF and Wnt3a intransgenic mice.

FIG. 59 Embryonic expression of GIPF and Wnt3a in the small and largeintestine of transgenic mice.

FIG. 60 Stabilization of β-catenin in transgenic mice that express GIPFand Wnt3a.

FIG. 61 H&E staining of sections of the small intestine of a transgenicmouse embryo that expresses GIPF and Wnt3a.

FIG. 62 A-K Diagrammatic representation of the construction of the RS-KOvector.

FIG. 63 Genomic map of wild type and recombinant RS-KO clones.

FIG. 64 A-K Diagrammatic representation of the construction of aknock-in vector pCk m4 KI for the expression of GIPF deletion mutant(SEQ ID NO: 91) in transgenic mice.

FIG. 65 A-C Diagrammatic representation of the construction of aknock-in vector pPS m4 KI for the expression of GIPF deletion mutant(SEQ ID NO: 91) in transgenic mice.

FIG. 66 Genomic map of wild type and recombinant Ck m4 KI clones.

FIG. 67 Genomic map of wild type and recombinant PS m4 KI clones.

FIG. 68 A-C Diagrammatic representation of the construction of aknock-in vector pCk VR KI for the expression of GIPF variant (SEQ ID NO:177; GenBank Accession Number AK098225) in transgenic mice.

FIG. 69 A-C Diagrammatic representation of the construction of aknock-in vector pPS VR KI for the expression of GIPF variant (SEQ ID NO:177; GenBank Accession Number AK098225) in transgenic mice.

FIG. 70 Genomic map of wild type and recombinant Ck VR KI clones.

FIG. 71 Genomic map of wild type and recombinant PS VR KI clones.

FIG. 72 Comparison of small and large intestines of control andtransgenic mice expressing the GIPF deletion mutant SEQ ID NO: 91.

FIG. 73 H&E staining of cross-sections of small intestine fromtransgenic mice expressing GIPF deletion mutant SEQ ID NO: 91 (lowmagnification).

FIG. 74 H&E staining of cross-sections of small intestine fromtransgenic mice expressing GIPF deletion mutant SEQ ID NO: 91 (highmagnification).

FIG. 75 Stabilization of Axin-2 in transgenic mice that express GIPFdeletion mutant SEQ ID NO: 91.

FIG. 76 Comparison of small and large intestines of control andtransgenic mice (PSVR KI) expressing GIPF variant (SEQ ID NO: 177;GenBank Accession Number AK098225) to that of a control animal.

FIG. 77 H&E staining of cross-sections of small intestine from controland transgenic mice (PSVR KI) expressing GIPF variant (SEQ ID NO: 177;GenBank Accession Number AK098225) (low magnification).

FIG. 78 H&E staining of cross-sections of small intestine from controland transgenic mice (PSVR KI) expressing GIPF variant (SEQ ID NO: 177;GenBank Accession Number AK098225) (high magnification).

FIG. 79 Stabilization of Axin-2 in control and transgenic mice thatexpress GIPF variant (SEQ ID NO: 177; GenBank Accession NumberAK098225).

FIG. 80 H&E staining of sections from the large intestine of controlanimal and an animal in which chronic IBD was induced by T-cell transfer(example 37).

4. DETAILED DESCRIPTION OF THE INVENTION

The polypeptides of the invention are depicted in FIG. 3, and aredescribed in detail below.

The GIPF polypeptide of SEQ ID NO: 4 is a 263-amino acid protein with apredicted molecular mass of approximately 29 kDa unglycosylated. SEQ IDNO:2 is a cDNA encoding GIPF polypeptide. The initial methionine startsat position 603 of SEQ ID NO: 2 and the putative stop codon begins atposition 1392 of SEQ ID NO: 2. Protein database searches with the BLASTalgorithm (Altschul S. F. et al., J. Mol. Evol. 36:290-300 (1993) andAltschul S. F. et al., J. Mol. Biol. 21:403-10 (1990), hereinincorporated by reference) indicate that SEQ ID NO: 4 is homologous toSEQ ID NO: 23 Stem Cell Growth Factor A-1 (SEQ ID NO: 10 from PCT WO01/77169 A2) (FIG. 4A), and human thrombospondin 1 (SEQ ID NO: 28) (FIG.4B).

A predicted approximately twenty-residue signal peptide (SEQ ID NO: 8)extends from residue 1 to residue 20 of SEQ ID NO: 4. The extracellularportion is useful on its own. The signal peptide region was predictedusing the Neural Network Signal P VI.I program (Nielsen et al., Int. J.Neural Syst. 8:581-599 (1997)), incorporated herein by reference) and/orusing Neural Network SignalP VI.I program (Nielsen et al, (1997) Int. J.Neural Syst. 8, 581-599). One of skill in the art will recognize thatthe actual cleavage site may be different than that predicted by thecomputer program. SEQ ID NO: 10 is the GIPF polypeptide of SEQ ID NO: 4that lacks the putative signal peptide (SEQ ID NO: 8).

Two species of polypeptides derived from SEQ ID NO: 4 have been clonedand purified in mammalian cell culture. SEQ ID NO: 10 is the polypeptideform purified from cellular medium of Chinese Hamster Ovary (CHO) cellsthat are transfected with a vector construct comprising nucleotidesequence of SEQ ID NO: 3. The polypeptide of SEQ ID NO: 10 is hereinknown as the dominant mature form of GIPF. SEQ ID NO: 9 is a nucleotidesequence that encodes the polypeptide of SEQ ID NO: 10. The N-terminalsequence for this polypeptide form was determined through Edmandegradation sequencing (Speicher, D. W. Methods 6: 248-261 (1994);Tempst et al., Methods 6: 248-261 (1994)). SEQ ID NO: 12 is the maturepolypeptide form isolated from the cellular medium of human embryonickidney 293 cells that are transfected with a vector construct comprisingSEQ ID NO: 3. SEQ ID NO: 11 is a corresponding nucleotide sequence thatencodes the polypeptide of SEQ ID NO: 12. Through Edman degradationsequencing, it has been determined that the polypeptide of SEQ ID NO: 12lacks the first 31 amino acid residues of SEQ ID NO: 4. The 31 aminoacid peptide comprises a consensus site (SEQ ID NO: 20) for furinprotease cleavage (Zhou et al., J Biol Chem 274:20745-20748 (1999),herein incorporated by reference in its entirety).

Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res.,Vol. 26(1) pp. 320-322 (1998) herein incorporated by reference) the GIPFpolypeptide (SEQ ID NO: 4) was examined for domains with homology toknown peptide domains. GIPF polypeptide of SEQ ID NO: 4 is expected tohave a thrombospondin type 1 domain (SEQ ID NO: 14 encoded by thenucleotide sequence of SEQ ID NO 13). The Pfam score for thethrombospondin type 1 domain contained within SEQ ID NO: 4 is 0.0034,and is predicted to be from amino acid residue 151 through 206 of SEQ IDNO: 4. The thrombospondin domain may be useful on its own.

Other forms of GIPF include a polypeptide having the amino acid sequenceof SEQ ID NO:4 except that the valine at position 50 of SEQ ID NO:4 isreplaced by an isoleucine (GIPF-I; SEQ ID NO:______). Another form ofGIPF-I has the amino acid sequence of SEQ ID NO:10 except that thevaline at position 30 of SEQ ID NO:10 is replaced by an isoleucine (SEQID NO:______). A third form of GIPF-I has the amino acid sequence of SEQID NO:12 except that the valine at position 19 of SEQ ID NO:12 isreplaced by an isoleucine (SEQ ID NO:______). Yet another form of GIPFincludes the amino acid sequence common to SEQ ID NO:4 and SEQ IDNO:178. Thus, this polypeptide ahas the amino acid sequence of aminoacids 33-263 of SEQ ID NO:4 (SEQ ID NO:______).

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol., vol. 6, pp. 219-235 (1999), hereinincorporated by reference), GIPF polypeptide of SEQ ID NO: 4 is expectedto have domains outlined in the table below, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine:

SEQ ID Identification eMATRIX domain Amino acid NO: p value No. nameSequence (position) 24 8.63e−10 IPB001862A Membrane attackPAQCEMSEWSPWGP complex CS (145-160) components/perforin/ complement C925 9.03e−10 IPB002174A Furin-like cysteine GKRQRRISAEGSQACA rich regionKGCELCSEVNGCLKCS (26-57) 26 9.80e−08 IPB000433 ZZ Zinc fingerIEHCEACFSHNFCTKC signature KP (99-115)

In order to control the production of either the dominant mature or themature polypeptide form that was predominantly produced by CHO and/or293 cells (SEQ ID NO: 10 and SEQ ID NO: 12, respectively), syntheticconstructs have been made. SEQ ID NO: 16 is a nucleotide sequenceincluded in a vector system that results in the expression of apolypeptide (SEQ ID NO: 16) in which the predicted signal peptide (SEQID NO: 8) adjoins the predominant mature form produced in 293 cells (SEQID NO: 10). SEQ ID NO: 17 is a nucleotide construct produced bysite-directed mutagenesis (Weiner et al., Gene 126:35-41 (1993)) tocontain a mutation in the furin-protease cleavage consensus site (SEQ IDNO: 22). This mutation changes the first arginine (R) residue of SEQ IDNO: 20 to a glutamine (Q). The arginine to glutamine mutation enablesthe production of the dominant mature form of GIPF by 293 cells (SEQ IDNO: 10).

Thrombospondins are a family of extracellular matrix proteins that areinvolved in cell-cell and cell-matrix communication (Lawler et al.,Curr. Opin. Cell Bio. 12:634-640 (2000)). More than five differentthrombospondins are known with distinct patterns of tissue distribution.Some tissues like heart, cartilage, and brain express most of thethrombospondin gene products. Thrombospondin-1 is a major constituent ofblood platelets. Thrombospondin-1 appears to function at the cellsurface to bring together membrane proteins and cytokines and othersoluble factors. Membrane proteins that bind thrombospondin-1 includeintegrins, integrin-associated protein (CD47), CD36, proteoglycans.Transforming growth factor β (TGFfβ) and platelet-derived growth factoralso bind thrombospondin-1.

Thrombospondin-1 is a large protein with many distinct domains. Itcontains a globular domain at both amino and carboxy terminus, a regionof homology with procollagen, and three types of repeated sequencemotifs termed thrombospondin (TSP) type 1, type 2, and type 3 repeats.TSP1 repeats have been found in various different proteins including,complement components (C6, C7, C8A etc.) extracellular matrix proteinslike ADAMTS, mindin, axonal guidance molecule like F-spondinsemaphorins, and also SCO-spondin, and TRAP proteins of Plasmodium.

Thrombospondin type 1 (TSP1) repeat can activate TGFfβ epithelialtissues which are involved in regulation of cell growth,differentiation, adhesion, migration, and death. TSP1 is furtherinvolved in protein binding, heparin binding, cell attachment, neuriteoutgrowth, inhibition of proliferation, inhibition of angiogenesis, andactivation of apoptosis. TSP1 domains of Plasmodium circumsporozoite(CS) protein and TRAP proteins are implicated in salivary gland invasionby the sporozoite.

TSP1 sequences are characterized by conserved cysteines, closely spacedtryptophans, and a cluster of basic residues. Spatial configuration ofTSP1 sequences shows two β-sheet domains which are shown to bind heparin(Kilpelainen et al (200) J. Biol Chem. 275, 13564-13570, incorporatedherein by reference). A similar spatial fold has been described forheparin-binding growth associated molecule (HB-GAM). HB-GAM is identicalto mitogenic and neurite outgrowth-promoting protein pleitrophin;osteoblast specific factor-1; heparin-binding neurotrophic factor; andheparin affin regulatory peptide. Expression of HB-GAM was shown to beassociated with extracellular matrix of axonal tracts and synapses, andalso with basement membranes outside of brain and in the cartilagematrix. Recently, N-syndecan has been shown to be a receptor for HB-GAMin brain and has been suggested to play roles in regulation ofhippocampal long-term potentiation, a form of brain plasticityimplicated in memory and learning. Therefore, TSP1 containing proteinsmay act as growth promoters and may exhibit GIPF activities.

In addition, thrombospondin, synthesized in bone marrow and depositedwithin the extracellular matrix, functions as a cytoadhesion moleculefor primary pluripotent progenitor cells, as well as for hematopoieticprogenitor cells committed to erythroid, granulocytic, andmegakaryocytic lineages. Thus thrombospondins may be important in bloodcell development (Long and Dixit (1990) Blood 75, 2311-2318,incorporated herein by reference).

GIPF polypeptides and polynucleotides of the invention may be used toinduce proliferation or differentiation of gastrointestinal crypt cells.They may also be used in the treatment of conditions whereepithelialization is required, such as for the treatment ofgastrointestinal disorders including chemotherapy and radiationtherapy-induced mucositis, mucositis of the oropharynx, lips andesophagus, inflammatory bowel disease, and other conditions includingwounds, burns, ophthalmic disorders, and any disorder where stimulationof epithelial cell proliferation or regeneration is desired. Thepolynucleotides and polypeptides of the invention may further beutilized to generate new tissues and organs that may aid patients inneed of transplanted tissues.

4.1 DEFINITIONS

In describing the present invention the following terms will be employedand are intended to be defined as indicated below.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an” and “the” include plural references unless thecontext clearly dictates otherwise.

The term “GIPF” refers to the “gastrointestinal proliferative factor”that is particularly active on epithelial cells.

In accordance with the present invention, the term “GIPF protein(s)” or“GIPF polypeptide(s) refers to the full-length protein defined by aminoacids Met¹ to Ala²⁶³ (SEQ ID NO: 4), fragments and analogs thereof.

The term “full-length GIPF,” “long form of GIPF”, “wild type GIPF”, or“native GIPF” as used herein all refer to the polypeptide that contains263 amino acid residues (SEQ ID NO: 4), as shown in FIG. 1B.

The term “GIPFwt” or “hGIPF” refer to the human wild type, full-lengthGIPF polypeptide (SEQ ID NO: 4); the term “GIPFt” refers to theV5His6-tagged polypeptide of human GIPF (SEQ ID NO: 6); and “mGIPFt”refers to the V5His6-tagged GIPF from mouse (SEQ ID NO: 69).

The term “fragment” refers to a polypeptide derived from the native GIPFthat does not include the entire sequence of GIPF. Such a fragment maybe a truncated version of the full-length molecule, for example SEQ IDNO: 9, and 12, as well as an internally deleted polypeptide, for exampleSEQ ID NO: 16. A GIPF fragment may have GIPF bioactivity as determinedby the effect of GIPF on the proliferation of epithelial cells in vitroand/or in vivo, as described herein.

The term “analog” refers to derivatives of the reference molecule. Theanalog may retain biological activity, as described above. In general,the term “analog” refers to compounds having a native polypeptidesequence and structure with one or more amino acid additions,substitutions (generally conservative in nature) and/or deletions,relative to the native molecule, so long as the modifications do notdestroy activity. SEQ ID NO: 18 is an example of a GIPF analog.Preferably, the analog has at least the same biological activity as theparent molecule, and may even display enhanced activity over the parentmolecule. Methods for making polypeptide analogs are known in the art.Particularly preferred analogs include substitutions that areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic: aspartate and glutamate; (2) basic: lysine, arginine, histidine;(3) non-polar: alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar: glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids. For example, it is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, an aspartatewith a glutamate, a threonine with a serine, or a similar conservativereplacement of an amino acid with a structurally related amino acid willpreserve the biological activity of GIPF.

Guidance in determining which amino acid residues may be replaced, addedor deleted without abolishing activities of interest, may be found bycomparing the sequence of the particular polypeptide with that ofhomologous peptides and minimizing the number of amino acid sequencechanges made in regions of high homology (conserved regions) or byreplacing amino acids with consensus sequence.

Alternatively, recombinant analogs encoding these same or similarpolypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsin the polynucleotide sequence may be reflected in the polypeptide ordomains of other peptides added to the polypeptide to modify theproperties of any part of the polypeptide, to change characteristicssuch as ligand-binding affinities, interchain affinities, ordegradation/turnover rate.

Preferably, amino acid “substitutions” are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, i.e., conservative amino acid replacements.“Conservative” amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. “Insertions” or “deletions” are preferably in the rangeof about 1 to 20 amino acids, more preferably 1 to 10 amino acids. Thevariation allowed may be experimentally determined by systematicallymaking insertions, deletions, or substitutions of amino acids in apolypeptide molecule using recombinant DNA techniques and assaying theresulting recombinant variants for activity.

Alternatively, where alteration of function is desired, insertions,deletions or non-conservative alterations can be engineered to producealtered polypeptides. Such alterations can, for example, alter one ormore of the biological functions or biochemical characteristics of thepolypeptides of the invention. For example, such alterations may changepolypeptide characteristics such as ligand-binding affinities,interchain affinities, or degradation/turnover rate. Further, suchalterations can be selected so as to generate polypeptides that arebetter suited for expression, scale up and the like in the host cellschosen for expression. For example, cysteine residues can be deleted orsubstituted with another amino acid residue in order to eliminatedisulfide bridges.

The term “derivative” refers to polypeptides chemically modified by suchtechniques as ubiquitination, labeling (e.g., with radionuclides orvarious enzymes), covalent polymer attachment such as pegylation(derivatization with polyethylene glycol) and insertion or substitutionby chemical synthesis of amino acids such as ornithine, which do notnormally occur in human proteins.

The terms “polypeptide” and “protein” refer to a polymer of amino acidresidues and are not limited to a minimum length of the product. Theterms also include, unless otherwise indicated, modifications of thepolypeptide that do not change the sequence of amino acids, for example,glycosylated, acetylated and phosphorylated forms. A polypeptide orprotein, for purposes of the present invention, may be synthetically orrecombinantly produced, as well as isolated from natural sources.

By “purified” and “isolated” is meant, when referring to a polypeptideor polynucleotide, that the indicated molecule is present in thesubstantial absence of other biological macromolecules of the same type.The term “purified” as used herein preferably means at least 75% byweight, more preferably at least 85% by weight, more preferably still atleast 95% by weight, and most preferably at least 98% by weight, ofbiological macromolecules of the same type are present in the sample. Inone embodiment, the polynucleotide or polypeptide is purified such thatit constitutes at least 95% by weight of the indicated biologicalmacromolecules present but water, buffers, and other small molecules,especially molecules having a molecular weight of less than 1000daltons, can be present.

An “isolated polynucleotide which encodes a particular polypeptide”refers to a nucleic acid molecule which is substantially free of othernucleic acid molecules that do not encode the subject polypeptide;however, the molecule may include some additional bases or moietieswhich do not deleteriously affect the basic characteristics of thecomposition.

The term “naturally occurring polypeptide” refers to polypeptidesproduced by cells that have not been genetically engineered andspecifically contemplates various polypeptides arising frompost-translational modifications of the polypeptide including, but notlimited to, acetylation, carboxylation, glycosylation, phosphorylation,lipidation and acylation.

The term “translated protein coding portion” means a sequence whichencodes for the full length protein which may include any leadersequence or a processing sequence.

The term “dominant mature protein coding sequence” refers to a sequencewhich encodes a peptide or protein without any leader/signal sequence.The “dominant mature protein portion” refers to that portion of theprotein without the leader/signal sequence. The “mature” form refers toa GIPF polypeptide that lacks the leader/signal sequence and the furincleavage site. The peptide may have the leader sequence and/or the furincleavage site removed during processing in the cell or the protein mayhave been produced synthetically or using a polynucleotide only encodingfor the mature protein coding sequence. It is contemplated that themature or dominant mature protein portion may or may not include aninitial methionine residue. The initial methionine is often removedduring processing of the peptide.

The term “isolated” as used herein refers to a nucleic acid orpolypeptide separated from at least one other component (e.g., nucleicacid or polypeptide) present with the nucleic acid or polypeptide in itsnatural source. In one embodiment, the nucleic acid or polypeptide isfound in the presence of (if anything) only a solvent, buffer, ion, orother components normally present in a solution of the same. The terms“isolated” and “purified” do not encompass nucleic acids or polypeptidespresent in their natural source.

The term “recombinant,” when used herein to refer to a polypeptide orprotein, means that a polypeptide or protein is derived from recombinant(e.g., microbial, insect, or mammalian) expression systems. “Microbial”refers to recombinant polypeptides or proteins made in bacterial orfungal (e.g., yeast) expression systems. As a product, “recombinantmicrobial” defines a polypeptide or protein essentially free of nativeendogenous substances and unaccompanied by associated nativeglycosylation. Polypeptides or proteins expressed in most bacterialcultures, e.g., E. coli, will be free of glycosylation modifications;polypeptides or proteins expressed in yeast will have a glycosylationpattern in general different from those expressed in mammalian cells.

By a “recombinant polypeptide” is intended a polypeptide which has beenprepared by recombinant DNA techniques as described herein. In general,the gene coding for the desired polypeptide is cloned and then expressedin transformed organisms, as described farther below. The host organismexpresses the foreign gene to produce the polypeptide under expressionconditions. Alternatively, the promoter controlling expression of anendogenous polypeptide can be altered to render a recombinantpolypeptide.

The term “active” refers to those forms of the polypeptide that retainthe biologic and/or immunologic activities of any naturally occurringpolypeptide. According to the invention, the terms “biologically active”or “biological activity” refer to a protein or peptide havingstructural, regulatory or biochemical functions of a naturally occurringmolecule. Likewise “biologically active” or “biological activity” refersto the capability of the natural, recombinant or synthetic GIPF peptide,or any peptide thereof, to induce a specific biological response inappropriate animals or cells and to bind with specific antibodies.

The term “secreted” includes a protein that is transported across orthrough a membrane, including transport as a result of signal sequencesin its amino acid sequence when it is expressed in a suitable host cell.“Secreted” proteins include without limitation proteins secreted wholly(e.g., soluble proteins) or partially (e.g., receptors) from the cell inwhich they are expressed. “Secreted” proteins also include withoutlimitation proteins that are transported across the membrane of theendoplasmic reticulum. “Secreted” proteins are also intended to includeproteins containing non-typical signal sequences (e.g. Interleukin-1Beta, see Krasney, P. A. and Young, P. R. (1992) Cytokine 4(2):134-143)and factors released from damaged cells (e.g. Interleukin-1 ReceptorAntagonist, see Arend, W. P. et. al. (1998) Annu. Rev. Immunol.16:27-55)

The term “polynucleotide” or “nucleic acid molecule” as used hereinrefers to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. This term refers only to theprimary structure of the molecule and thus includes double- andsingle-stranded DNA and RNA. It also includes known types ofmodifications, for example, labels which are known in the art,methylation, “caps”, substitution of one or more of the naturallyoccurring nucleotides with an analog, internucleotide modifications suchas, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) andwith charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.), those containing pendant moieties, such as, for example proteins(including for e.g., nucleases, toxins, antibodies, signal peptides,poly-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelates (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide. Generally, nucleicacid segments provided by this invention may be assembled from fragmentsof the genome and short oligonucleotide linkers, or from a series ofoligonucleotides, or from individual nucleotides, to provide a syntheticnucleic acid which is capable of being expressed in a recombinanttranscriptional unit comprising regulatory elements derived from amicrobial or viral operon, or a eukaryotic gene.

The terms “oligonucleotide fragment” or a “polynucleotide fragment”,“portion,” or “segment” or “probe” or “primer” are used interchangeablyand refer to a sequence of nucleotide residues which are at least about5 nucleotides, more preferably at least about 7 nucleotides, morepreferably at least about 9 nucleotides, more preferably at least about11 nucleotides and most preferably at least about 17 nucleotides. Thefragment is preferably less than about 500 nucleotides, preferably lessthan about 200 nucleotides, more preferably less than about 100nucleotides, more preferably less than about 50 nucleotides and mostpreferably less than 30 nucleotides. Preferably the probe is from about6 nucleotides to about 200 nucleotides, preferably from about 15 toabout 50 nucleotides, more preferably from about 17 to 30 nucleotidesand most preferably from about 20 to 25 nucleotides. Preferably thefragments can be used in polymerase chain reaction (PCR), varioushybridization procedures or microarray procedures to identify or amplifyidentical or related parts of mRNA or DNA molecules. A fragment orsegment may uniquely identify each polynucleotide sequence of thepresent invention. Preferably the fragment comprises a sequencesubstantially similar to a portion of SEQ ID NO: 1, 2, 5, 7, 9, 11, 13,15, 17, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104 or 177.

Probes may, for example, be used to determine whether specific mRNAmolecules are present in a cell or tissue or to isolate similar nucleicacid sequences from chromosomal DNA as described by Walsh et al. (Walsh,P. S. et al., 1992, PCR Methods Appl 1:241-250). They may be labeled bynick translation, Klenow fill-in reaction, PCR, or other methods wellknown in the art. Probes of the present invention, their preparationand/or labeling are elaborated in Sambrook, J. et al., 1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; orAusubel, F. M. et al., 1989, Current Protocols in Molecular Biology,John Wiley & Sons, New York N.Y., both of which are incorporated hereinby reference in their entirety.

The nucleic acid sequences of the present invention also include thesequence information from any of the nucleic acid sequences of SEQ IDNO: 1, 2, 5, 7, 9, 11, 13, 15, 17, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104 or 177. The sequence information can be a segment of SEQ ID NO:1, 2, 5, 7, 9, 11, 13, 15, 17, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104 or 177 that uniquely identifies or represents the sequenceinformation of SEQ ID NO: 1, 2, 5, 7, 9, 11, 13, 15, 17, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104 or 177. One such segment can be atwenty-mer nucleic acid sequence because the probability that atwenty-mer is fully matched in the human genome is 1 in 300. In thehuman genome, there are three billion base pairs in one set ofchromosomes. Because 4²⁰ possible twenty-mers exist, there are 300 timesmore twenty-mers than there are base pairs in a set of humanchromosomes. Using the same analysis, the probability for aseventeen-mer to be fully matched in the human genome is approximately 1in 5. When these segments are used in arrays for expression studies,fifteen-mer segments can be used. The probability that the fifteen-meris fully matched in the expressed sequences is also approximately one infive because expressed sequences comprise less than approximately 5% ofthe entire genome sequence.

Similarly, when using sequence information for detecting a singlemismatch, a segment can be a twenty-five mer. The probability that thetwenty-five mer would appear in a human genome with a single mismatch iscalculated by multiplying the probability for a full match (1÷4²⁵) timesthe increased probability for mismatch at each nucleotide position(3×25). The probability that an eighteen mer with a single mismatch canbe detected in an array for expression studies is approximately one infive. The probability that a twenty-mer with a single mismatch can bedetected in a human genome is approximately one in five.

The term “open reading frame,” ORF, means a series of nucleotidetriplets coding for amino acids without any termination codons and is asequence translatable into protein.

The terms “operably linked” or “operably associated” refer tofunctionally related nucleic acid sequences. For example, a promoter isoperably associated or operably linked with a coding sequence if thepromoter controls the transcription of the coding sequence. Whileoperably linked nucleic acid sequences can be contiguous and in the samereading frame, certain genetic elements e.g. repressor genes are notcontiguously linked to the coding sequence but still controltranscription/translation of the coding sequence.

The terms “recombinant DNA molecule,” or “recombinant polynucleotide”are used herein to refer to a polynucleotide of genomic, cDNA,semisynthetic, or synthetic origin which, by virtue of its origin ormanipulation: (1) is not associated with all or a portion of apolynucleotide with which it is associated in nature, (2) is linked to apolynucleotide other than that to which it is linked in nature, or (3)does not occur in nature. Thus, the term encompasses “syntheticallyderived” nucleic acid molecules.

The terms “complementary” or “complementarity” refer to the naturalbinding of polynucleotides by base pairing. For example, the sequence5′-AGT-3′ binds to the complementary sequence 3′-TCA-5′. Complementaritybetween two single-stranded molecules may be “partial” such that onlysome of the nucleic acids bind or it may be “complete” such that totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between the nucleic acid strands has significanteffects on the efficiency and strength of the hybridization between thenucleic acid strands.

The term “stringent” is used to refer to conditions that are commonlyunderstood in the art as stringent. Stringent conditions can includehighly stringent conditions (i.e., hybridization to filter-bound DNA in0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C.), and moderately stringentconditions (i.e., washing in 0.2×SSC/0.1% SDS at 42° C.). Otherexemplary hybridization conditions are described herein in the examples.

In instances of hybridization of deoxyoligonucleotides, additionalexemplary stringent hybridization conditions include washing in6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-baseoligonucleotides), 48° C. (for 17-base oligonucleotides), 55° C. (for20-base oligonucleotides), and 60° C. (for 23-base oligonucleotides).

As used herein, “substantially equivalent” can refer both to nucleotideand amino acid sequences, for example a mutant sequence, that variesfrom a reference sequence by one or more substitutions, deletions, oradditions, the net effect of which does not result in an adversefunctional dissimilarity between the reference and subject sequences.Typically, such a substantially equivalent sequence varies from one ofthose listed herein by no more than about 35% (i.e., the number ofindividual residue substitutions, additions, and/or deletions in asubstantially equivalent sequence, as compared to the correspondingreference sequence, divided by the total number of residues in thesubstantially equivalent sequence is about 0.35 or less). Such asequence is said to have 65% sequence identity to the listed sequence.In one embodiment, a substantially equivalent, e.g., mutant, sequence ofthe invention varies from a listed sequence by no more than 30% (70%sequence identity); in a variation of this embodiment, by no more than25% (75% sequence identity); and in a further variation of thisembodiment, by no more than 20% (80% sequence identity) and in a furthervariation of this embodiment, by no more than 10% (90% sequenceidentity) and in a further variation of this embodiment, by no more that5% (95% sequence identity). Substantially equivalent, e.g., mutant,amino acid sequences according to the invention preferably have at least80% sequence identity with a listed amino acid sequence, more preferablyat least 90% sequence identity. Substantially equivalent nucleotidesequence of the invention can have lower percent sequence identities,taking into account, for example, the redundancy or degeneracy of thegenetic code. Preferably, nucleotide sequence has at least about 65%identity, more preferably at least about 75% identity, and mostpreferably at least about 95% identity. For the purposes of the presentinvention, sequences having substantially equivalent biological activityand substantially equivalent expression characteristics are consideredsubstantially equivalent. For the purposes of determining equivalence,truncation of the mature sequence (e.g., via a mutation which creates aspurious stop codon) should be disregarded. Sequence identity may bedetermined, e.g., using the Jotun Hein method (Hein, J. (1990) MethodsEnzymol. 183:626-645). Identity between sequences can also be determinedby other methods known in the art, e.g. by varying hybridizationconditions.

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. The term“expression vector” includes plasmids, cosmids or phages capable ofsynthesizing the GIPF protein encoded by the respective recombinant genecarried by the vector. Preferred vectors are those capable of autonomousreplication and expression of nucleic acids to which they are linked.

The term “transformation” means introducing DNA into a suitable hostcell so that the DNA is replicable, either as an extrachromosomalelement, or by chromosomal integration.

The term “transfection” refers to the taking up of an expression vectorby a suitable host cell, whether or not any coding sequences are in factexpressed. The term “infection” refers to the introduction of nucleicacids into a suitable host cell by use of a virus or viral vector.

The term “transcriptional regulatory elements” and transcriptionalregulatory sequences” are used interchangeably to refer to DNA sequencesnecessary for the expression of an operably linked coding sequence in aparticular organism. The control sequences that are suitable forprokaryotes, for example, include a promoter, optionally an operatorsequence, and a ribosome binding site. Eukaryotic cells are known toutilize promoters, enhancers, splicing signals and polyadenylationsignals. These terms are intended to encompass all elements that promoteor regulate transcription, including promoters, core elements requiredfor basic interaction of RNA polymerase and transcription factors,upstream elements, enhancers, and response elements (Lewin, “Genes V”(Oxford University Press, Oxford) pages 847-873).

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then optionallytrans-RNA spliced and translated into the protein encoded by the codingsequence.

The term “tissue-specific promoter” means a nucleotide sequence thatserves as a promoter, i.e. regulates expression of a selected DNAsequence operably linked to the promoter, and which effects theexpression of the selected DNA sequence in specific cells, such asB-cells. In an illustrative embodiment, gene constructs utilizing B-cellspecific promoters can be used to preferentially direct expression of aGIPF protein or protein fragment in B-cells.

The term “expression modulating fragment,” EMF, means a series ofnucleotides that modulates the expression of an operably linked ORF oranother EMF.

As used herein, a sequence is said to “modulate the expression of anoperably linked sequence” when the expression of the sequence is alteredby the presence of the EMF. EMFs include, but are not limited to,promoters, and promoter modulating sequences (inducible elements). Oneclass of EMFs is nucleic acid fragments which induce the expression ofan operably linked ORF in response to a specific regulatory factor orphysiological event.

The term “recombinant expression vehicle or vector” refers to a plasmidor phage or virus or vector, for expressing a polypeptide from a DNA(RNA) sequence. An expression vehicle can comprise a transcriptionalunit comprising an assembly of (1) a genetic element or elements havinga regulatory role in gene expression, for example, promoters orenhancers, (2) a structural or coding sequence which is transcribed intomRNA and translated into protein, and (3) appropriate transcriptioninitiation and termination sequences. Structural units intended for usein yeast or eukaryotic expression systems preferably include a leadersequence enabling extracellular secretion of translated protein by ahost cell. Alternatively, where recombinant protein is expressed withouta leader or transport sequence, it may include an amino terminalmethionine residue. This residue may or may not be subsequently cleavedfrom the expressed recombinant protein to provide a final product.

The term “recombinant expression system” means host cells which havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit extrachromosomally.Recombinant expression systems as defined herein will expressheterologous polypeptides or proteins upon induction of the regulatoryelements linked to the DNA segment or synthetic gene to be expressed.This term also means host cells which have stably integrated arecombinant genetic element or elements having a regulatory role in geneexpression, for example, promoters or enhancers. Recombinant expressionsystems as defined herein will express polypeptides or proteinsendogenous to the cell upon induction of the regulatory elements linkedto the endogenous DNA segment or gene to be expressed. The cells can beprokaryotic or eukaryotic.

The term “transgene” refers to a nucleic acid sequence which is partlyor entirely heterologous i.e. foreign, to the transgenic animal or cellinto which it is introduced, or, is homologous to an endogenous gene ofthe transgenic animal or cell into which it is introduced, but which isdesigned to be inserted, or is inserted into the animal's genome in sucha way as to alter the genome of the cell into which it is inserted. e.g.it is inserted at a location which differs from that of the naturalgene). A transgene can be operably linked to one or more transcriptionalregulatory sequences and any other nucleic acids, such as introns, thatmay be necessary for optimal expression of a selected nucleic acid.

Accordingly, a “transgene construct” refers to a nucleic acid whichincludes a transgene, and optionally such other nucleic acid sequencesas transcriptionally regulatory sequences, polyadenylation sites,replication origins, marker genes etc. which may be useful in thegeneral manipulation of the transgene for insertion in the genome of ahost organism.

The term “transgenic” is used herein as an adjective to describe theproperty, for example, of an animal or construct, of harboring atransgene. For instance, as used herein, a “transgenic organism” is anyanimal, preferably a non-human mammal, in which one or more of the cellsof the animal contain heterologous nucleic acids introduced by way ofhuman intervention, such as by transgenic techniques known in the art.The nucleic acid is introduced into the cell, directly, or indirectly byintroduction into a precursor of the cell, by way of a deliberategenetic manipulation, such as by microinjection or by infection with arecombinant virus. The nucleic acid may be integrated within achromosome, or it may be extrachromosomally replicating DNA. In thetransgenic animals described herein, the transgene causes cells toexpress or overexpress GIPF proteins.

The term “pluripotent” refers to the capability of a cell todifferentiate into a number of differentiated cell types that arepresent in an adult organism. A pluripotent cell is restricted in itsdifferentiation capability in comparison to a totipotent cell.

The term “embryonic stem cells (ES)” refers to a cell that can give riseto many differentiated cell types in an embryo or an adult, includingthe germ cells. The term “germ line stem cells (GSCs)” refers to stemcells derived from primordial stem cells that provide a steady andcontinuous source of germ cells for the production of gametes. The term“primordial germ cells (PGCs)” refers to a small population of cells setaside from other cell lineages particularly from the yolk sac,mesenteries, or gonadal ridges during embryogenesis that have thepotential to differentiate into germ cells and other cells. PGCs are thesource from which GSCs and ES cells are derived. The PGCs, the GSCs andthe ES cells are capable of self-renewal. Thus these cells not onlypopulate the germ line and give rise to a plurality of terminallydifferentiated cells that comprise the adult specialized organs, but areable to regenerate themselves. The term “totipotent” refers to thecapability of a cell to differentiate into all of the cell types of anadult organism. The term “pluripotent” refers to the capability of acell to differentiate into a number of differentiated cell types thatare present in an adult organism. A pluripotent cell is restricted inits differentiation capability in comparison to a totipotent cell.

The terms “founder line” and “founder animal” refer to those animalsthat are the mature product of the embryos to which the transgene wasadded i.e. those animals that grew from the embryos into which DNA wasinserted and that were implanted into one or more surrogate hosts.

The terms “progeny” and “progeny of the transgenic animal” refer to anyand all offspring of every generation subsequent to the originallytransformed mammals.

The term “non-human mammal” refers to all members of the class Mammaliaexcept humans. “Mammal” refers tot any animal classified as a mammal,including humans, domestic and farm animals, and zoo, sports, or petanimals, such as a mouse, rat, rabbit, pig, sheep, goat, cattle andhigher primates.

The terms “treat” or “treatment” refer to both therapeutic andprophylactic or preventative measures, wherein the object is to preventor lessen an undesired physiological change or condition, such aschemotherapy or radiation therapy-induced mucositis. For the purposes ofthis invention, beneficial or desired clinical results include, but arenot limited to alleviation of symptoms, diminishment of extent of thedisease, stabilized state of the disease, whether detectable orundetectable.

A “disorder” is any condition that would benefit from treatment with amolecule identified using the transgenic animal model of the invention.This includes chronic and acute disorders or diseases including thosepathological conditions which predispose the mammal to the disorder inquestion. Non-limiting examples of disorders to be treated hereininclude mucositis, inflammatory bowel disease and skin lesions. Apreferred disorder to be treated in accordance with the presentinvention is mucositis.

“Inflammatory bowel disease (IBD)” herein refers to idiopathic orchronic inflammatory disease of either or both the small intestine andlarge bowel, and includes Crohn's disease, ulcerative colitis, IBDcaused by infectious agents, and antibiotic associated IBD.

“Mucositis” herein refers to inflammation of the mucous membranes of thealimentary tract including the oropharynx and lips, esophagus, and largeand small intestine.

“Short Bowel Syndrome” or “SBS” herein refers to a condition ofnutritional malabsorption resulting from anatomical or functional lossof a significant length of the small intestine.

The terms “effective amount” or “pharmaceutically effective amount”refer to a nontoxic but sufficient amount of the agent to provide thedesired biological result. That result can be reduction and/oralleviation of the signs, symptoms, or causes of a disease, or any otherdesired alteration of a biological system. For example, an effectiveamount of a GIPF fragment for use with the present methods is an amountsufficient to stimulate epithelial cell stimulation or proliferation,and preferably an amount sufficient to cause increased regeneration ofthe gastrointestinal epithelium in a subject suffering from chemotherapyor radiation therapy-induced mucositis, inflammatory bowel disease, orother disorders where epithelial cell proliferation is desired. Suchamounts are described below. An appropriate “effective” amount in anyindividual case may be determined by one of ordinary skill in the artusing routine experimentation.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual without causingany undesirable biological effects or interacting in a deleteriousmanner with any of the components of the composition in which it iscontained.

By “physiological pH” or a “pH in the physiological range” is meant a pHin the range of approximately 7.0 to 8.0 inclusive. Preferredphysiological pH is in the range of approximately 7.2 to 7.6 inclusive.

As used herein, the term “subject” encompasses mammals and non-mammals.Examples of mammals include, but are not limited to, any member of theMammalia class: humans, non-human primates such as chimpanzees, andother apes and monkey species; farm animals such as cattle, horses,sheep, goats, swine; domestic animals such as rabbits, dogs, and cats;laboratory animals including rodents, such as rats, mice and guineapigs, and the like. Examples of non-mammals include, but are not limitedto, birds, fish and the like. The term does not denote a particular ageor gender.

4.2 COMPOSITIONS OF THE INVENTION

4.2.1 Nucleic Acid Compositions

The invention is based on the discovery that compositions comprising theepithelial cell growth factor polypeptide, GIPF, and the polynucleotidesencoding the GIPF polypeptide stimulate the growth and proliferation ofintestinal epithelial cells including crypt cells. Therefore, the use ofthese compositions for the diagnosis and treatment of conditions whereinstimulation of epithelial cell proliferation or regeneration is desired,is contemplated.

The isolated polynucleotides of the invention include, but are notlimited to a polynucleotide comprising any of the nucleotide sequencesof SEQ ID NO: 2, 3, 5, 7, 9, 11, 13, 15, 17, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104 or 177; a polynucleotide comprising the full lengthprotein coding sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 84,86, 88, 90, 92, 94, 96, 98, 100, 102, 104 or 177; (for example codingfor SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105 or 178); and a polynucleotide comprising thenucleotide sequence encoding the mature and dominant mature proteincoding sequence of the polypeptide of SEQ ID NO: 4. The polynucleotidesof the present invention also include, but are not limited to, apolynucleotide that hybridizes under stringent conditions to (a) thecomplement of any of the nucleotides sequences of SEQ ID NO: 2, 3, 5, 7,9, 11, 13, 15, 17, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104 or 177;(b) a polynucleotide encoding any one of the polypeptides of SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,105 or 178; (c) a polynucleotide which is an allelic variant of anypolynucleotides recited above; (d) a polynucleotide which encodes aspecies homolog of any of the proteins recited above; or (e) apolynucleotide that encodes a polypeptide comprising a specific domainor truncation of the polypeptides of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16,18, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or 178. Domains ofinterest include extracellular, transmembrane, or cytoplasmic domains,or combinations thereof; and catalytic and substrate binding domains.

The polynucleotides of the invention include naturally occurring orwholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA,e.g., mRNA. The polynucleotides may include all of the coding region ofthe cDNA or may represent a portion of the coding region of the cDNA.

The present invention also provides compositions comprising genescorresponding to the cDNA sequences disclosed herein. The correspondinggenes can be isolated in accordance with known methods using thesequence information disclosed herein. Such methods include thepreparation of probes or primers from the disclosed sequence informationfor identification and/or amplification of genes in appropriate genomiclibraries or other sources of genomic materials. Further 5′ and 3′sequence can be obtained using methods known in the art. For example,full length cDNA or genomic DNA that corresponds to any of thepolynucleotide of SEQ ID NO: 2 can be obtained by screening appropriatecDNA or genomic DNA libraries under suitable hybridization conditionsusing any of the polynucleotides of SEQ ID NO: 2, 3, 5, 7, 9, 11, 13,15, 17, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104 or 177 or aportion thereof as a probe. Alternatively, the polynucleotides of SEQ IDNO: 2, 3, 5, 7, 9, 11, 13, 15, 17, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104 or 177 may be used as the basis for suitable primer(s) thatallow identification and/or amplification of genes in appropriategenomic DNA or cDNA libraries.

The nucleic acid sequences of the invention can be assembled from ESTsand sequences (including cDNA and genomic sequences) obtained from oneor more public databases, such as dbEST, gbpri, and UniGene. The ESTsequences can provide identifying sequence information, representativefragment or segment information, or novel segment information for thefull-length gene.

The polynucleotides of the invention also provide polynucleotidesincluding nucleotide sequences that are substantially equivalent to thepolynucleotides recited above. Polynucleotides according to theinvention can have, e.g., at least about 65%, at least about 70%, atleast about 75%, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, or 89%, more typically at least about 90%, 91%, 92%, 93%, or 94%and even more typically at least about 95%, 96%, 97%, 98% or 99%sequence identity to a polynucleotide recited above.

Included within the scope of the nucleic acid sequences of the inventionare nucleic acid sequence fragments that hybridize under stringentconditions to any of the nucleotide sequences of SEQ ID NO: 1, 6, 8, 10,12, 14, 17, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104 or 177, orcomplements thereof, which fragment is greater than about 5 nucleotides,preferably 7 nucleotides, more preferably greater than 9 nucleotides andmost preferably greater than 17 nucleotides. Fragments of, e.g. 15, 17,or 20 nucleotides or more that are selective for (i.e. specificallyhybridize to any one of the polynucleotides of the invention) arecontemplated. Probes capable of specifically hybridizing to apolynucleotide can differentiate polynucleotide sequences of theinvention from other polynucleotide sequences in the same family ofgenes or can differentiate human genes from genes of other species, andare preferably based on unique nucleotide sequences.

The sequences falling within the scope of the present invention are notlimited to these specific sequences, but also include allelic andspecies variations thereof. Allelic and species variations can beroutinely determined by comparing the sequence provided in SEQ ID NO: 2,3, 5, 7, 9, 11, 13, 15, 17, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104 or 177, a representative fragment thereof, or a nucleotide sequenceat least 90% identical, preferably 95% identical, to SEQ ID NO: 2, 3, 5,7, 9, 11, 13, 15, 17, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104 or177 with a sequence from another isolate of the same species.Furthermore, to accommodate codon variability, the invention includesnucleic acid molecules coding for the same amino acid sequences as dothe specific ORFs disclosed herein. In other words, in the coding regionof an ORF, substitution of one codon for another codon that encodes thesame amino acid is expressly contemplated.

The nearest neighbor result for the nucleic acids of the presentinvention can be obtained by searching a database using an algorithm ora program. Preferably, a BLAST which stands for Basic Local AlignmentSearch Tool is used to search for local sequence alignments (Altshul, S.F. J Mol. Evol. 36 290-300 (1993) and Altschul S. F. et al. J. Mol.Biol. 21:403-410 (1990))

Species homologs (or orthologs) of the disclosed polynucleotides andproteins are also provided by the present invention. Species homologsmay be isolated and identified by making suitable probes or primers fromthe sequences provided herein and screening a suitable nucleic acidsource from the desired species.

The invention also encompasses allelic variants of the disclosedpolynucleotides or proteins; that is, naturally-occurring alternativeforms of the isolated polynucleotide which also encodes proteins whichare identical, homologous or related to that encoded by thepolynucleotides.

The nucleic acid sequences of the invention are further directed tosequences which encode analogs of the described nucleic acids. Theseamino acid sequence analogs may be prepared by methods known in the artby introducing appropriate nucleotide changes into a native or variantpolynucleotide. There are two variables in the construction of aminoacid sequence variants: the location of the mutation and the nature ofthe mutation. Nucleic acids encoding the amino acid sequence analogs arepreferably constructed by mutating the polynucleotide to encode an aminoacid sequence that does not occur in nature. These nucleic acidalterations can be made at sites that differ in the nucleic acids fromdifferent species (variable positions) or in highly conserved regions(constant regions). Sites at such locations will typically be modifiedin series, e.g., by substituting first with conservative choices (e.g.,hydrophobic amino acid to a different hydrophobic amino acid) and thenwith more distant choices (e.g., hydrophobic amino acid to a chargedamino acid), and then deletions or insertions may be made at the targetsite. Amino acid sequence deletions generally range from about 1 to 30residues, preferably about 1 to 10 residues, and are typicallycontiguous. Amino acid insertions include amino- and/orcarboxyl-terminal fusions ranging in length from one to one hundred ormore residues, as well as intrasequence insertions of single or multipleamino acid residues. Intrasequence insertions may range generally fromabout 1 to 10 amino residues, preferably from 1 to 5 residues. Examplesof terminal insertions include the heterologous signal sequencesnecessary for secretion or for intracellular targeting in different hostcells and sequences such as poly-histidine sequences useful forpurifying the expressed protein.

In a preferred method, polynucleotides encoding the novel amino acidsequences are changed via site-directed mutagenesis. This method usesoligonucleotide sequences to alter a polynucleotide to encode thedesired amino acid variant, as well as sufficient adjacent nucleotideson both sides of the changed amino acid to form a stable duplex oneither side of the site being changed. In general, the techniques ofsite-directed mutagenesis are well known to those of skill in the artand this technique is exemplified by publications such as, Edelman etal., DNA 2:183 (1983). A versatile and efficient method for producingsite-specific changes in a polynucleotide sequence was published byZoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982). PCR may alsobe used to create amino acid sequence variants of the novel nucleicacids. When small amounts of template DNA are used as starting material,primer(s) that differs slightly in sequence from the correspondingregion in the template DNA can generate the desired amino acid variant.PCR amplification results in a population of product DNA fragments thatdiffer from the polynucleotide template encoding the polypeptide at theposition specified by the primer. The product DNA fragments replace thecorresponding region in the plasmid and this gives a polynucleotideencoding the desired amino acid variant.

A further technique for generating amino acid variants is the cassettemutagenesis technique described in Wells et al., Gene 34:315 (1985); andother mutagenesis techniques well known in the art, such as, forexample, the techniques in Sambrook et al., supra, and Current Protocolsin Molecular Biology, Ausubel et al. Due to the inherent degeneracy ofthe genetic code, other DNA sequences which encode substantially thesame or a functionally equivalent amino acid sequence may be used in thepractice of the invention for the cloning and expression of these novelnucleic acids. Such DNA sequences include those which are capable ofhybridizing to the appropriate novel nucleic acid sequence understringent conditions.

Polynucleotides encoding preferred polypeptide truncations of theinvention can be used to generate polynucleotides encoding chimeric orfusion proteins comprising one or more domains of the invention andheterologous protein sequences.

The polynucleotides of the invention additionally include the complementof any of the polynucleotides recited above. The polynucleotide can beDNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods andalgorithms for obtaining such polynucleotides are well known to those ofskill in the art and can include, for example, methods for determininghybridization conditions that can routinely isolate polynucleotides ofthe desired sequence identities.

In accordance with the invention, polynucleotide sequences comprisingthe dominant mature or mature protein coding sequences, coding for anyone of SEQ ID NO: 6 or 8, or functional equivalents thereof, may be usedto generate recombinant DNA molecules that direct the expression of thatnucleic acid, or a functional equivalent thereof, in appropriate hostcells. Also included are the cDNA inserts of any of the clonesidentified herein.

A polynucleotide according to the invention can be joined to any of avariety of other nucleotide sequences by well-established recombinantDNA techniques (see Sambrook J et al. (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, NY). Useful nucleotidesequences for joining to polynucleotides include an assortment ofvectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids,and the like, that are well known in the art. Accordingly, the inventionalso provides a vector including a polynucleotide of the invention and ahost cell containing the polynucleotide. In general, the vector containsan origin of replication functional in at least one organism, convenientrestriction endonuclease sites, and a selectable marker for the hostcell. Vectors according to the invention include expression vectors,replication vectors, probe generation vectors, and sequencing vectors. Ahost cell according to the invention can be a prokaryotic or eukaryoticcell and can be a unicellular organism or part of a multicellularorganism.

The present invention further provides recombinant constructs comprisinga nucleic acid having any of the nucleotide sequences of SEQ ID NO: 2,3, 5, 7, 9, 11, 13, 15, 17, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104 or 177 or a fragment thereof or any other GIPF polynucleotides. Inone embodiment, the recombinant constructs of the present inventioncomprise a vector, such as a plasmid or viral vector, into which anucleic acid having any of the nucleotide sequences of SEQ ID NO: 2, 3,5, 7, 9, 11, 13, 15, 17, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104or 177 or a fragment thereof is inserted. In the case of a vectorcomprising one of the ORFs of the present invention, the vector mayfurther comprise regulatory sequences, including for example, apromoter, operably linked to the ORF. Large numbers of suitable vectorsand promoters are known to those of skill in the art and arecommercially available for generating the recombinant constructs of thepresent invention. The following vectors are provided by way of example.Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a,pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG(Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). In one embodiment,the nucleic acid of SEQ ID NO: 3 is inserted in the CKP2KI vector of theinvention as described in the examples.

The isolated polynucleotide of the invention may be operably linked toan expression control sequence such as the pMT2 or pED expressionvectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490(1991), in order to produce the protein recombinantly. Many suitableexpression control sequences are known in the art. General methods ofexpressing recombinant proteins are also known and are exemplified in R.Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein“operably linked” means that the isolated polynucleotide of theinvention and an expression control sequence are situated within avector or cell in such a way that the protein is expressed by a hostcell which has been transformed (transfected) with the ligatedpolynucleotide/expression control sequence.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lac, lacZ, T3, T7, gpt, lambda PR, and trc.Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-1.Selection of the appropriate vector and promoter is well within thelevel of ordinary skill in the art. Generally, recombinant expressionvectors will include origins of replication and selectable markerspermitting transformation of the host cell, e.g., the ampicillinresistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoterderived from a highly expressed gene to direct transcription of adownstream structural sequence. Such promoters can be derived fromoperons encoding glycolytic enzymes such as 3-phosphoglycerate kinase(PGK), a-factor, acid phosphatase, or heat shock proteins, among others.The heterologous structural sequence is assembled in appropriate phasewith translation initiation and termination sequences, and preferably, aleader sequence capable of directing secretion of translated proteininto the periplasmic space or extracellular medium. Optionally, theheterologous sequence can encode a fusion protein including an aminoterminal identification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct. Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed. Followingtransformation of a suitable host strain and growth of the host strainto an appropriate cell density, the selected promoter is induced orderepressed by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. Cells aretypically harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification.

In addition to the use of expression vectors in the practice of thepresent invention, the present invention further includes novelexpression vectors comprising promoter elements operatively linked topolynucleotide sequences encoding a protein of interest. One example ofsuch a vector is the pcDNA/vector, which is described in Example 8.

4.2.2 Hosts

The present invention further provides host cells genetically engineeredwith the vectors of this invention, which may be, for example, a cloningvector or an expression vector that contain the polynucleotides of theinvention. For example, such host cells may contain nucleic acids of theinvention introduced into the host cell using known transformation,transfection or infection methods. The vector may be, for example, inthe form of a plasmid, a viral particle, a phage etc. The engineeredhost cells can be cultured in conventional nutrient media modified asappropriate for activating promoters, selecting transformants oramplifying GIPF genes. The culture conditions, such as temperature, pH,and the like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan. Thepresent invention still further provides host cells geneticallyengineered to express the polynucleotides of the invention, wherein suchpolynucleotides are in operative association with a regulatory sequenceheterologous to the host cell which drives expression of thepolynucleotides in the cell.

The host cell can be a higher eukaryotic host cell, such as a mammaliancell, a lower eukaryotic host cell, such as a yeast cell, or the hostcell can be a prokaryotic cell, such as a bacterial cell. Introductionof the recombinant construct into the host cell can be effected bycalcium phosphate transfection, DEAE, dextran mediated transfection, orelectroporation (Davis, L. et al., Basic Methods in Molecular Biology(1986)). The host cells containing one of polynucleotides of theinvention, can be used in conventional manners to produce the geneproduct encoded by the isolated fragment (in the case of an ORF) or canbe used to produce a heterologous protein under the control of the EMF.

Any host/vector system can be used to express one or more of the GIPFpolypeptides. These include, but are not limited to, eukaryotic hostssuch as HeLa cells, Cv-1 cell, COS cells, and Sf9 cells, as well asprokaryotic host such as E. coli and B. subtilis. The most preferredcells are those which do not normally express the particular polypeptideor protein or which expresses the polypeptide or protein at low naturallevel. Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al., inMolecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989), the disclosure of which is hereby incorporated byreference.

Various mammalian cell culture systems can be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, for example, the C127, 3T3, CHO, HeLa and BHK cell tines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter, and also any necessary ribosome binding sites,polyadenylation site, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking nontranscribed sequences. DNAsequences derived from the SV40 viral genome, for example, SV40 origin,early promoter, enhancer, splice, and polyadenylation sites may be usedto provide the required nontranscribed genetic elements. Recombinantpolypeptides and proteins produced in bacterial culture are usuallyisolated by initial extraction from cell pellets, followed by one ormore salting-out, aqueous ion exchange or size exclusion chromatographysteps. Protein refolding steps can be used, as necessary, in completingconfiguration of the mature protein. Finally, high performance liquidchromatography (HPLC) can be employed for final purification steps.Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents.

A number of types of cells may act as suitable host cells for expressionof the protein. Mammalian host cells include, for example, monkey COScells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1cells, other transformed primate cell lines, normal diploid cells, cellstrains derived from in vitro culture of primary tissue, primaryexplants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkatcells. Preferably, GIPF proteins are expressed in Chinese Hamster Ovary(CHO) cells, and human embryonic kidney 293 cells.

Alternatively, it may be possible to produce the protein in lowereukaryotes such as yeast or in prokaryotes such as bacteria. Potentiallysuitable yeast strains include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, Pichiapastoris or any yeast strain capable of expressing heterologousproteins. Potentially suitable bacterial strains include Escherichiacoli, Bacillus subtilis, Salmonella typhimurium, or any bacterial straincapable of expressing heterologous proteins. If the protein is made inyeast or bacteria, it may be necessary to modify the protein producedtherein, for example by phosphorylation or glycosylation of theappropriate sites, in order to obtain the functional protein. Suchcovalent attachments may be accomplished using known chemical orenzymatic methods.

4.2.3 Chimeric and Fusion Proteins

The invention also provides GIPF chimeric or fusion proteins. As usedherein, a GIPF “chimeric protein” or “fusion protein” comprises a GIPFpolypeptide operatively-linked to a non-GIPF polypeptide. A “GIPFpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a GIPF protein, whereas a “non-GIPF polypeptide” refersto a polypeptide having an amino acid sequence corresponding to aprotein that is not substantially homologous to the GIPF protein, e.g.,a protein that is different from the GIPF protein and that is derivedfrom the same or a different organism. Within a GIPF fusion protein theGIPF polypeptide can correspond to all or a portion of a GIPF protein.In one embodiment, a GIPF fusion protein comprises at least onebiologically active portion of a GIPF protein. In another embodiment, aGIPF fusion protein comprises at least two biologically active portionsof a GIPF protein. In yet another embodiment, a GIPF fusion proteincomprises at least three biologically active portions of a GIPF protein.Within the fusion protein, the term “operatively-linked” is intended toindicate that the GIPF polypeptide and the non-GIPF polypeptide arefused in-frame with one another. The non-GIPF polypeptide can be fusedto the N-terminus or C-terminus of the GIPF polypeptide.

In one embodiment, the fusion protein is a GST-GIPF fusion protein inwhich the GIPF sequences are fused to the C-terminus of the GST(glutathione S-transferase) sequences. Such fusion proteins canfacilitate the purification of recombinant GIPF polypeptides. In anotherembodiment, the fusion protein is a GIPF protein containing aheterologous signal sequence at its N-terminus. In certain host cells(e.g., mammalian host cells), expression and/or secretion of GIPF can beincreased through use of a heterologous signal sequence. Preferably, theGIPF polypeptide is fused with a V5-His tag for easy detection with ananti-V5 antibody and for rapid purification as described in theexamples.

A GIPF chimeric or fusion protein of the invention can be produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, e.g., by employing blunt-endedor stagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley & Sons, 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A GIPF-encoding nucleic acid can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to theGIPF protein.

4.2.4 Polypeptide Compositions

The pharmaceutical compositions of the invention comprise isolated GIPFpolypeptides that include, but are not limited to, a polypeptidecomprising: the amino acid sequence set forth as any one of SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,105 or 178, or an amino acid sequence encoded by any one of thenucleotide sequences SEQ ID NO: 2, 3, 5, 7, 9, 11, 13, 15, 17, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104 or 177. Polypeptides of theinvention also include polypeptides preferably with biological orimmunological activity that are encoded by: (a) a polynucleotide havingany one of the nucleotide sequences set forth in SEQ ID NO: 2, 3, 5, 7,9, 11, 13, 15, 17, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, or 104 or(b) polynucleotides encoding any one of the amino acid sequences setforth as SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 85, 87, 89, 91, 93, 95,97, 99, 101, 103, 105 or 178 or (c) polynucleotides that hybridize tothe complement of the polynucleotides of either (a) or (b) understringent hybridization conditions. The invention also providesbiologically active or immunologically active variants of any of theamino acid sequences set forth as SEQ ID NO: 4, 6, 8, 10, 12, 14, 16,18, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or 178; and“substantial equivalents” thereof (e.g., with at least about 65%, atleast about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, or 89%, more typically at least about 90%, 91%,92%, 93%, or 94% and even more typically at least about 95%, 96%, 97%,98% or 99%, most typically at least about 99% amino acid identity) thatretain biological activity. Polypeptides encoded by allelic variants mayhave a similar, increased, or decreased activity compared topolypeptides comprising SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, 105 or 178.

Fragments of the proteins of the present invention which are capable ofexhibiting biological activity are also encompassed by the presentinvention. Fragments of the protein may be in linear form or they may becyclized using known methods, for example, as described in H. U.Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S.McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both ofwhich are incorporated herein by reference. Such fragments may be fusedto carrier molecules such as immunoglobulins for many purposes,including increasing the valency of protein binding sites.

The present invention also provides both full-length and dominant matureforms (for example, without a signal sequence or precursor sequence) ormature forms (for example, lacking the signal sequence and the furincleavage site) of the disclosed proteins. The protein coding sequence isidentified in the sequence listing by translation of the disclosednucleotide sequences. The mature form of such protein may be obtained byexpression of a full-length polynucleotide in a suitable mammalian cellor other host cell. The sequence of the mature form of the protein isalso determinable from the amino acid sequence of the full-length form.

Protein compositions of the present invention may further comprise anacceptable carrier, such as a hydrophilic, e.g., pharmaceuticallyacceptable, carrier.

The present invention further provides isolated polypeptides encoded bythe nucleic acid fragments of the present invention or by degeneratevariants of the nucleic acid fragments of the present invention. By“degenerate variant” is intended nucleotide fragments which differ froma nucleic acid fragment of the present invention (e.g., an ORF) bynucleotide sequence but, due to the degeneracy of the genetic code,encode an identical polypeptide sequence. Preferred nucleic acidfragments of the present invention are the ORFs that encode proteins.

A variety of methodologies known in the art can be utilized to obtainany one of the isolated polypeptides or proteins of the presentinvention. At the simplest level, the amino acid sequence can besynthesized using commercially available peptide synthesizers. Thesynthetically-constructed protein sequences, by virtue of sharingprimary, secondary or tertiary structural and/or conformationalcharacteristics with proteins may possess biological properties incommon therewith, including protein activity. This technique isparticularly useful in producing small peptides and fragments of largerpolypeptides. Fragments are useful, for example, in generatingantibodies against the native polypeptide. Thus, they may be employed asbiologically active or immunological substitutes for natural, purifiedproteins in screening of therapeutic compounds and in immunologicalprocesses for the development of antibodies.

The polypeptides and proteins of the present invention can alternativelybe purified from cells which have been altered to express the desiredpolypeptide or protein. As used herein, a cell is said to be altered toexpress a desired polypeptide or protein when the cell, through geneticmanipulation, is made to produce a polypeptide or protein which itnormally does not produce or which the cell normally produces at a lowerlevel. One skilled in the art can readily adapt procedures forintroducing and expressing either recombinant or synthetic sequencesinto eukaryotic or prokaryotic cells in order to generate a cell whichproduces one of the polypeptides or proteins of the present invention.

The invention also relates to methods for producing a polypeptidecomprising growing a culture of host cells of the invention in asuitable culture medium, and purifying the protein from the cells or theculture in which the cells are grown. For example, the methods of theinvention include a process for producing a polypeptide in which a hostcell containing a suitable expression vector that includes apolynucleotide of the invention is cultured under conditions that allowexpression of the encoded polypeptide. The polypeptide can be recoveredfrom the culture, conveniently from the culture medium, or from a lysateprepared from the host cells and further purified. Preferred embodimentsinclude those in which the protein produced by such process is a fulllength or mature form of the protein.

In an alternative method, the polypeptide or protein is purified frombacterial cells which are transformed with GIPF-encoding DNA to producethe polypeptide or protein. One skilled in the art can readily followknown methods for isolating polypeptides and proteins in order to obtainone of the isolated polypeptides or proteins of the present invention.These include, but are not limited to, immunochromatography, HPLC,size-exclusion chromatography, ion-exchange chromatography, andimmuno-affinity chromatography. See, e.g., Scopes, Protein Purification:Principles and Practice, Springer-Verlag (1994); Sambrook, et al., inMolecular Cloning: A Laboratory Manual; Ausubel et al., CurrentProtocols in Molecular Biology. Polypeptide fragments that retainbiological/immunological activity include fragments comprising greaterthan about 100 amino acids, or greater than about 200 amino acids, andfragments that encode specific protein domains.

The purified polypeptides can be used in in vitro binding assays whichare well known in the art to identify molecules which bind to thepolypeptides. These molecules include but are not limited to, for e.g.,small molecules, molecules from combinatorial libraries, antibodies orother proteins. The molecules identified in the binding assay are thentested for antagonist or agonist activity in in vivo tissue culture oranimal models that are well known in the art. In brief, the moleculesare titrated into a plurality of cell cultures or animals and thentested for either cell/animal death or prolonged survival of theanimal/cells.

The protein of the invention may also be expressed as a product oftransgenic animals, e.g., as a component of the milk of transgenic cows,goats, pigs, or sheep which are characterized by somatic or germ cellscontaining a nucleotide sequence encoding the protein.

The proteins provided herein also include proteins characterized byamino acid sequences similar to those of purified proteins but intowhich modification are naturally provided or deliberately engineered.For example, modifications, in the peptide or DNA sequence, can be madeby those skilled in the art using known techniques. Modifications ofinterest in the protein sequences may include the alteration,substitution, replacement, insertion or deletion of a selected aminoacid residue in the coding sequence. For example, one or more of thecysteine residues may be deleted or replaced with another amino acid toalter the conformation of the molecule. Techniques for such alteration,substitution, replacement, insertion or deletion are well known to thoseskilled in the art (see, e.g., U.S. Pat. No. 4,518,584). Preferably,such alteration, substitution, replacement, insertion or deletionretains the desired activity of the protein. Regions of the protein thatare important for the protein function can be determined by variousmethods known in the art including the alanine-scanning method whichinvolved systematic substitution of single or strings of amino acidswith alanine, followed by testing the resulting alanine-containingvariant for biological activity. This type of analysis determines theimportance of the substituted amino acid(s) in biological activity.Regions of the protein that are important for protein function may bedetermined by the eMATRIX program.

Other fragments and derivatives of the sequences of proteins which wouldbe expected to retain protein activity in whole or in part and areuseful for screening or other immunological methodologies may also beeasily made by those skilled in the art given the disclosures herein.Such modifications are encompassed by the present invention.

The protein may also be produced by operably linking the isolatedpolynucleotide of the invention to suitable control sequences in one ormore insect expression vectors, and employing an insect expressionsystem. Materials and methods for baculovirus/insect cell expressionsystems are commercially available in kit form from, e.g., Invitrogen,San Diego, Calif., U.S.A. (the MaxBat™ kit), and such methods are wellknown in the art, as described in Summers and Smith, Texas AgriculturalExperiment Station Bulletin No. 1555 (1987), incorporated herein byreference. As used herein, an insect cell capable of expressing apolynucleotide of the present invention is “transformed.”

The protein of the invention may be prepared by culturing transformedhost cells under culture conditions suitable to express the recombinantprotein. The resulting expressed protein may then be purified from suchculture (i.e., from culture medium or cell extracts) using knownpurification processes, such as gel filtration and ion exchangechromatography. The purification of the protein may also include anaffinity column containing agents which will bind to the protein; one ormore column steps over such affinity resins as concanavalin A-agarose,Heparin-Toyopearl™ or Cibacrom blue 3GA Sepharose™; one or more stepsinvolving hydrophobic interaction chromatography using such resins asphenyl ether, butyl ether, or propyl ether; or immunoaffinitychromatography.

Alternatively, the protein of the invention may also be expressed in aform which will facilitate purification. For example, it may beexpressed as a fusion protein, such as those of maltose binding protein(MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), or as a Histag. Kits for expression and purification of such fusion proteins arecommercially available from New England BioLab (Beverly, Mass.),Pharmacia (Piscataway, N.J.) and Invitrogen, respectively. The proteincan also be tagged with an epitope and subsequently purified by using aspecific antibody directed to such epitope. One such epitope (“FLAG®”)is commercially available from Kodak (New Haven, Conn.).

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the protein. Some or all of the foregoingpurification steps, in various combinations, can also be employed toprovide a substantially homogeneous isolated recombinant protein. Theprotein thus purified is substantially free of other mammalian proteinsand is defined in accordance with the present invention as an “isolatedprotein.”

The polypeptides of the invention include GIPF analogs. This embracesfragments of GIPF polypeptide, as well as GIPF polypeptides whichcomprise one or more amino acids deleted, inserted, or substituted.Also, analogs of the GIPF polypeptide of the invention embrace fusionsof the GIPF polypeptides or modifications of the GIPF polypeptides,wherein the GIPF polypeptide or analog is fused to another moiety ormoieties, e.g., targeting moiety or another therapeutic agent. Suchanalogs may exhibit improved properties such as activity and/orstability. Examples of moieties which may be fused to the GIPFpolypeptide or an analog include, for example, targeting moieties whichprovide for the delivery of polypeptide to the small intestine, e.g.,antibodies to the small intestine, or antibodies to receptor and ligandsexpressed on gastrointestinal cells. Other moieties which may be fusedto GIPF polypeptide include therapeutic agents which are used fortreatment, for example cytokines or other medications, ofgastrointestinal disorders, and other conditions as recited herein.

4.2.5 Gene Therapy

The invention provides gene therapy to treat the diseases cited herein.Delivery of a functional gene encoding polypeptides of the invention toappropriate cells is effected ex vivo, in situ, or in vivo by use ofvectors, and more particularly viral vectors (e.g., adenovirus,adeno-associated virus, or a retrovirus), or ex vivo by use of physicalDNA transfer methods (e.g., liposomes or chemical treatments). See, forexample, Anderson, Nature, supplement to vol. 392, no. 6679, pp. 25-20(1998). For additional reviews of gene therapy technology see Friedmann,Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84(1990); and Miller, Nature, 357: 455-460 (1992).

As discussed above, a “vector” is any means for the transfer of anucleic acid according to the invention into a host cell. Preferredvectors are viral vectors, such as retroviruses, herpes viruses,adenoviruses and adeno-associated viruses. Thus, a gene or nucleic acidsequence encoding a GIPF protein or polypeptide domain fragment thereofis introduced in vivo, ex vivo, or in vitro using a viral vector orthrough direct introduction of DNA. Expression in targeted tissues canbe effected by targeting the transgenic vector to specific cells, suchas with a viral vector or a receptor ligand, or by using atissue-specific promoter, or both.

Viral vectors commonly used for in vivo or ex vivo targeting and therapyprocedures are DNA-based vectors and retroviral vectors. Methods forconstructing and using viral vectors are known in the art [see, e.g.,Miller and Rosman, BioTechniques 7:980-990 (1992)]. Preferably, theviral vectors are replication defective, that is, they are unable toreplicate autonomously in the target cell. In general, the genome of thereplication defective viral vectors which are used within the scope ofthe present invention lack at least one region which is necessary forthe replication of the virus in the infected cell. These regions caneither be eliminated (in whole or in part), be rendered non-functionalby any technique known to a person skilled in the art. These techniquesinclude the total removal, substitution (by other sequences, inparticular by the inserted nucleic acid), partial deletion or additionof one or more bases to an essential (for replication) region. Suchtechniques may be performed in vitro (on the isolated DNA) or in situ,using the techniques of genetic manipulation or by treatment withmutagenic agents. Preferably, the replication defective virus retainsthe sequences of its genome which are necessary for encapsulating theviral particles.

DNA viral vectors include an attenuated or defective DNA virus, such asbut not limited to herpes simplex virus (HSV), papillomavirus,Epstein-Barr virus (EBV), adenovirus, adeno-associated virus (AAV), andthe like. Defective viruses, which entirely or almost entirely lackviral genes, are preferred. Defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Thus, a specific tissue can bespecifically targeted. Examples of particular vectors include, but arenot limited to, a defective herpes virus 1 (HSV1) vector [Kaplitt etal., Molec. Cell. Neurosci. 2:320-330 (1991)], defective herpes virusvector lacking a glyco-protein L gene [Patent Publication RD 371005 A],or other defective herpes virus vectors [International PatentPublication No. WO 94/21807, published Sep. 29, 1994; InternationalPatent Publication No. WO 92/05263, published Apr. 2, 1994]; anattenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al. [J. Clin. Invest. 90:626-630 (1992); seealso La Salle et al., Science 259:988-990 (1993)]; and a defectiveadeno-associated virus vector [Samulski et al., J. Virol. 61:3096-3101(1987); Samulski et al., J. Virol. 63:3822-3828 (1989); Lebkowski etal., Mol. Cell. Biol. 8:3988-3996 (1988)].

Preferably, for in vivo administration, an appropriate immunosuppressivetreatment is employed in conjunction with the viral vector, e.g.,adenovirus vector, to avoid immuno-deactivation of the viral vector andtransfected cells. For example, immunosuppressive cytokines, such asinterleukin-12 (IL-12), interferon-γ (IFN-γ), or anti-CD4 antibody, canbe administered to block humoral or cellular immune responses to theviral vectors [see, e.g., Wilson, Nature Medicine (1995)]. In addition,it is advantageous to employ a viral vector that is engineered toexpress a minimal number of antigens.

In a preferred embodiment, the vector is an adenovirus vector. As shownin the Examples, the adenovirus vector has shown itself to beparticularly effective for delivery of the GIPF polypeptide, as shown bythe unexpectedly efficient effects of stimulating intestinal epithelialcell proliferation resulting in marked, diffuse thickening of the mucosaby crypt epithelial hyperplasia and a marked increase in crypt lengthand complex branching. Adenoviruses are eukaryotic DNA viruses that canbe modified to efficiently deliver a nucleic acid of the invention to avariety of cell types. Various serotypes of adenovirus exist. Of theseserotypes, preference is given, within the scope of the presentinvention, to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5)or adenoviruses of animal origin (see WO94/26914). Those adenoviruses ofanimal origin which can be used within the scope of the presentinvention include adenoviruses of canine, bovine, murine (example: May1, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian, andsimian (example: SAV) origin.

Preferably, the replication defective adenoviral vectors of theinvention comprise the ITRs, an encapsidation sequence and the nucleicacid of interest. Still more preferably, at least the E1 region of theadenoviral vector is non-functional. Other regions may also be modified,in particular the E3 region (WO95/02697), the E2 region (WO94/28938),the E4 region (WO94/28152, WO94/12649 and WO95/02697), or in any of thelate genes L1-L5.

In a preferred embodiment, the adenoviral vector has a deletion in theE1 and E3 region. Examples of E1-deleted adenoviruses are disclosed inEP 185,573, the contents of which are incorporated herein by reference.

The replication defective recombinant adenoviruses according to theinvention can be prepared by any technique known to the person skilledin the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham,EMBO J. 3 (1984) 2917). In particular, they can be prepared byhomologous recombination between an adenovirus and a plasmid whichcarries, inter alia, the DNA sequence of interest. The homologousrecombination is effected following cotransfection of the saidadenovirus and plasmid into an appropriate cell line. The cell linewhich is employed should preferably (i) be transformable by the saidelements, and (ii) contain the sequences which are able to complementthe part of the genome of the replication defective adenovirus,preferably in integrated form in order to avoid the risks ofrecombination. Examples of cell lines which may be used are the humanembryonic kidney cell line 293 (Graham et al., J. Gen. Virol. 36 (1977)59) which contains the left-hand portion of the genome of an Ad5adenovirus (12%) integrated into its genome, and cell lines which areable to complement the E1 and E4 functions, as described in applicationsWO94/26914 and WO95/02697. Recombinant adenoviruses are recovered andpurified using standard molecular biological techniques, which are wellknown to one of ordinary skill in the art.

Promoters that may be used in the present invention include bothconstitutive promoters and regulated (inducible) promoters. The promotermay be naturally responsible for the expression of the nucleic acid. Itmay also be from a heterologous source. In particular, it may bepromoter sequences of eukaryotic or viral genes. For example, it may bepromoter sequences derived from the genome of the cell which it isdesired to infect. Likewise, it may be promoter sequences derived fromthe genome of a virus, including the adenovirus used. In this regard,there may be mentioned, for example, the promoters of the E1A, MLP, CMVand RSV genes and the like.

In addition, the promoter may be modified by addition of activating orregulatory sequences or sequences allowing a tissue-specific orpredominant expression (enolase and GFAP promoters and the like).Moreover, when the nucleic acid does not contain promoter sequences, itmay be inserted, such as into the virus genome downstream of such asequence.

Some promoters useful for practice of this invention are ubiquitouspromoters (e.g., HPRT, vimentin, actin, tubulin), intermediate filamentpromoters (e.g., desmin, neurofilaments, keratin, GFAP), therapeuticgene promoters (e.g., MDR type, CFTR, factor VIII), tissue-specificpromoters (e.g., actin promoter in smooth muscle cells), promoters whichare preferentially activated in dividing cells, promoters which respondto a stimulus (e.g., steroid hormone receptor, retinoic acid receptor),tetracycline-regulated transcriptional modulators, cytomegalovirusimmediate-early, retroviral LTR, metallothionein, SV-40, E1a, and MLPpromoters. Tetracycline-regulated transcriptional modulators and CMVpromoters are described in WO 96/01313, U.S. Pat. Nos. 5,168,062 and5,385,839, the contents of which are incorporated herein by reference.

Thus, the promoters which may be used to control gene expressioninclude, but are not limited to, the cytomegalovirus (CMV) promoter, theSV40 early promoter region (Benoist and Chambon, 1981, Nature290:304-310), the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1441-1445), the regulatory sequences of the metallothioneingene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expressionvectors such as the b-lactamase promoter (Villa-Kamaroff, et al., 1978,Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter(DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also“Useful proteins from recombinant bacteria” in Scientific American,1980, 242:74-94; promoter elements from yeast or other fungi such as theGal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK(phosphoglycerol kinase) promoter, alkaline phosphatase promoter; andthe animal transcriptional control regions, which exhibit tissuespecificity and have been utilized in transgenic animals: elastase Igene control region which is active in pancreatic acinar cells (Swift etal., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring HarborSymp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515);insulin gene control region which is active in pancreatic beta cells(Hanahan, 1985, Nature 315:115-122), immunoglobulin gene control regionwhich is active in lymphoid cells (Grosschedl et al., 1984, Cell38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al.,1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus controlregion which is active in testicular, breast, lymphoid and mast cells(Leder et al., 1986, Cell 45:485-495), albumin gene control region whichis active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276),alpha-fetoprotein gene control region which is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science235:53-58), alpha 1-antitrypsin gene control region which is active inthe liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globingene control region which is active in myeloid cells (Mogram et al.,1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94), myelinbasic protein gene control region which is active in oligodendrocytecells in the brain (Readhead et al., 1987, Cell 48:703-712), myosinlight chain-2 gene control region which is active in skeletal muscle(Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormonegene control region which is active in the hypothalamus (Mason et al.,1986, Science 234:1372-1378).

Introduction of any one of the nucleotides of the present invention or agene encoding the polypeptides of the present invention can also beaccomplished with extrachromosomal substrates (transient expression) orartificial chromosomes (stable expression). Cells may also be culturedex vivo in the presence of proteins of the present invention in order toproliferate or to produce a desired effect on or activity in such cells.Treated cells can then be introduced in vivo for therapeutic purposes.In addition to the use of viral vectors in the practice of the presentinvention, the present invention further includes a novel vectorcomprising operator and promoter elements operatively linked topolynucleotide sequences encoding a protein of interest. The noveladenoviral vector is the pAdenoVator-CMV5-Intron vector, which isdescribed in detail in Examples.

4.2.6 Transgenic Animals

The polynucleotides of the present invention also make possible thedevelopment of chimeric animals that specifically express GIPFpolypeptides in B cells. Such animals are useful as models for studyingthe in vivo activities of polypeptide as well as for studying modulatorsof the polypeptides of the invention. A preferred embodiment of theinvention relates to a transgenic knock-in (KI) mouse model that wasdesigned to determine the biological function of GIPF in a rapid manner.The transgenic KI animal model is described in International ApplicationPCT/JP02/11236, and published as WO2003/041495. The transgenic modelrelates to a GIPF transgene that encodes the B-cell specific expressionof GIPF under the control of the immunoglobulin kappa light chainpromoter. The transgene is introduced into TT2F ES cells, which containintact immunoglobulin heavy and light chain loci, and the ES cells thatcontain the GIPF transgene are implanted into mice that lack bothalleles for the antibody heavy chain (IgH-KOAH^(−/−)) (Kitamura et al.,Nature 350:423-426 (1991), herein incorporated by reference in itsentirety). Thus, the expression of the immunoglobulin kappa light chainscan only occur in functional B cells that are derived from the ES cellsthat express the IG chains (WO 00/10383; EP 1106061A1). Similarly, theexpression of GIPF by B-cells occurs only in the GIPF-KI chimeric mice.The transgenic animal model of the invention thus allows for a speedyphenotypic analysis of chimeric animals, rather than heterozygous orhomozygous animals containing transgenes transmitted through thegermline. In addition, the expression of the transgene is restricted toB cells, which secrete the GIPF protein into the animal's circulation,thus exposing every tissue to GIPF, and allowing for a rapid assessmentof the biological effect of GIPF, or any other encoded polypeptide. Itis intended that the transgenic system of the present invention can beused for expressing and assessing the biological function of anypolypeptide. Another advantage of the transgenic model of the inventionrelates to the temporal expression of the transgene. The activity of thekappa light chain promoter begins at approximately 14 dayspost-gestation and the remarkable elevation of circulatingimmunoglobulin concentration is observed after weaning, thus avoidingany potentially deleterious effects that GIPF might have on the earlydevelopment of the mouse. An exemplary embodiment of the transgenicanimal of the invention is described in the Examples.

4.2.6.1 General Method of Making Transgenic Non-Human Mammals

The transgenic animals of the present invention all include within aplurality of their cells a transgene of the present invention, whichtransgene alters the phenotype of the “host cell” with respect to thespecific expression of GIPF by B cells, which secrete GIPF polypeptidesinto the circulation of the transgenic animal. Various aspects oftransgenic animal technology are well known in the art, and aredescribed in detail in literature, such as Hogan et al., Manipulatingthe Mouse Embryo (Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., [1986]). Although the making of transgenic animals is illustratedherein with reference to transgenic mice, this is only for illustrativepurpose, and is not to be construed as limiting the scope of theinvention. This specific disclosure can be readily adapted by thoseskilled in the art to incorporate GIPF transgene sequences into anynon-human mammal utilizing the methods and materials described below.Animals suitable for transgenic experiments can be obtained fromstandard commercial sources such as Taconic (Germantown, N.Y.).

A. Transgene Construct

Construction of transgenes can be accomplished using any suitablegenetic engineering techniques well known in the art, including, withoutlimitation, the standard techniques of restriction endonucleasedigestion, ligation, transformation, plasmid purification, DNAsequencing etc as described in Sambrook et al., Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory, N.Y., (1989)).

The transgenes of the present invention are typically operably linked totranscriptional regulatory sequences, such as promoters and/orenhancers, to regulate expression of the transgene in a particularmanner. In certain embodiments, the useful transcriptional regulatorysequences are those that are highly regulated with respect to activity,both temporally and spatially. Thus, the promoters of choice can bethose that are active only in particular tissues or cell types.Promoters/enhancers which may be used to control the expression of thetransgene in vivo include, but are not limited to, the humancytomegalovirus (CMV) promoter/enhancer (Karasuyama et al., J. Exp. Med.169: 13 [1989]), the human β-actin promoter (Gunning et al., Proc. Natl.Acad. Sci. USA 84: 4831-4835 [1987]), the glucocorticoid-induciblepromoter present in the mouse mammary tumor virus long terminal repeat(MMTV LTR) (Kiessig et al., Mol. Cell. Biol. 4: 1354-1362 [1984]), thelong terminal repeat sequences of Moloney murine leukemia virus (MuLVLTR) (Weiss et al. [1985] RNA Tumor Viruses, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.), the SV40 early or late regionpromoter (Benoist et al. Nature 290: 304-310 [1981]; Templeton et al.Mol. Cell. Biol., 4: 817 [1984]; and Sprague et al., J. Virol., 45: 773[1983]), the promoter contained in the 3′ long terminal repeat of Roussarcoma virus (RSV) (Yamamoto et al., Cell 22: 787-797 [1980]), theherpes simplex virus (HSV) thymidine kinase promoter/enhancer (Wagner etal. Proc. Natl. Acad. Sci. USA 82: 3567-71 [1981]), metallothionein (MT)promoter (Palmiter et al., Nature 300: 611-615 [1982]), and the herpessimplex virus LAT promoter (Wolfe et al. Nature Genetics 1: 379-384[1992]). Preferably the promoter is the P2 promoter of theimmunoglobulin kappa light chain (REF).

In addition to the transgene and the transcriptional regulatorysequence, the vectors useful for preparing the transgenes of thisinvention typically contain one or more other elements useful foroptimizing expression of the transgene in the host animal. Thus, thetransgene construct may include transcription termination elements, suchas to direct polyadenylation of an mRNA transcript, as well as intronicsequences. For example, the transgene can be flanked at its 3′ end bySV40 sequences (SV40 intron/pA) which add the transcription terminationand polyadenylation signals to the transgene transcript. In yet otherembodiments, the transgene can include intron sequences. In manyinstances, the expression of a transgene is increased by the presence ofone or more introns in the coding sequence.

In still other embodiments, the transgene construct may includeadditional elements which facilitate its manipulation in cells (e.g., inbacterial cells) prior to insertion in the intended recipient cell. Forinstance, the vector may include origin of replication elements foramplification in prokaryotic cells. Moreover, the transgene constructmay contain selectable markers for isolating cells, either from therecipient animal, or cells generated as intermediate in making thetransgenic animal (i.e., bacterial cells used for amplifying theconstruct or ES cells used for introducing the transgene). Selectablemarker genes may encode proteins necessary for the survival and/orgrowth of transfected cells under selective culture conditions. Typicalselection marker genes encode proteins that, for example: (i) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline or kanamycin for prokaryotic host cells, and neomycin,hygromycin or methotrexate for mammalian cells; or (ii) complementauxotrophic deficiencies of the cell.

B. Cells Used for Introduction of Transgene

In an exemplary embodiment, the “transgenic non-human mammals” of theinvention are produced by introducing GIPF transgene into the germlineof the non-human mammal. Embryonal target cells at various developmentalstages can be used to introduce GIPF transgene. Different methods areused depending on the stage of development of the embryonal target cell.The specific line(s) of any animal used to practice this invention areselected for general good health, good embryo yields, good pronuclearvisibility in the embryo, and good reproductive fitness.

In one embodiment, the transgene construct is introduced into a singlestage embryo. Generally, the female animals are superovulated by hormonetreatment, mated and fertilized eggs are recovered. For example, in caseof mice, females six weeks of age are induced to superovulate with a 5IU injection (0.1 ml, i.p.) of pregnant mare serum gonadotropin (PMSG;Sigma) followed 48 hours later by a 5 IU injection (0.1 ml, i.p.) ofhuman chorionic gonadotropin (hCG; Sigma). FVB strain of mice are usedin this case. Females are then mated immediately with a stud maleovernight. Such females are next examined for copulation plugs. Thosethat have mated are euthanized by CO₂ asphyxiation or cervicaldislocation and embryos are recovered from excised oviducts and placedin Dulbecco's phosphate buffered saline with 0.5% bovine serum albumin(BSA; Sigma). Surrounding cumulus cells are removed with hyaluronidase(1 mg/ml). Pronuclear embryos are then washed and placed in Earle'sbalanced salt solution containing 0.5% BSA (EBSS) in a 37.5° C.incubator with a humidified atmosphere at 5% CO₂, 95% air until the timeof injection.

Normally, fertilized embryos are incubated in suitable media until thepronuclei appear. At about this time, the transgene is introduced intothe female or male pronucleus as described below. In some species suchas mice, the male pronucleus is preferred. For example, the exogenousgenetic material is added to the early male pronucleus, as soon aspossible after the formation of the male pronucleus, which is when themale and female pronuclei are well separated and both are located closeto the cell membrane. Alternatively, the exogenous genetic materialcould be added to the nucleus of the sperm after it has been induced toundergo decondensation. Sperm containing the exogenous genetic materialcan then be added to the ovum or the decondensed sperm could be added tothe ovum with the transgene constructs being added as soon as possiblethereafter.

In addition to similar biological considerations, physical ones alsogovern the amount (e.g., volume) of exogenous genetic material, whichcan be added to the nucleus of the zygote, or to the genetic materialwhich forms a part of the zygote nucleus. Generally, the volume ofexogenous genetic material inserted will not exceed about 10 picoliters.The physical effects of addition must not be so great as to physicallydestroy the viability of the zygote. The biological limit of the numberand variety of DNA sequences will vary depending upon the particularzygote and functions of the exogenous genetic material and will bereadily apparent to one skilled in the art, because the geneticmaterial, including the exogenous genetic material, of the resultingzygote must be biologically capable of initiating and maintaining thedifferentiation and development of the zygote into a functionalorganism.

The number of copies of the transgene constructs which are added to thezygote is dependent upon the total amount of exogenous genetic materialadded and will be the amount which enables the genetic transformation tooccur. Theoretically only one copy is required; however, generally,numerous copies are utilized, for example, 1,000-20,000 copies of thetransgene construct, in order to insure that one copy is functional.

C. Methods of Introducing Transgene

Each transgene construct to be inserted into the cell must first be inthe linear form since the frequency of recombination is higher withlinear molecules of DNA as compared to the circular molecules.Therefore, if the construct has been inserted into a vector,linearization is accomplished by digesting the DNA with a suitablerestriction endonuclease selected to cut only within the vector sequenceand not within the transgene sequence.

Introduction of the transgene into the embryo may be accomplished by anymeans known in the art so long as it is not destructive to the cell,nuclear membrane or other existing cellular or genetic structures. Someof the widely used methods include microinjection, electroporation, orlipofection. Following introduction of the transgene, the embryo may beincubated in vitro for varying amounts of time, or reimplanted into thesurrogate host, or both. One common method is to incubate the embryos invitro for about 1-7 days, depending on the species, and then reimplantthem into the surrogate host.

The zygote is the best target for introducing the transgene construct bymicroinjection method. In the mouse, the male pronucleus reaches thesize of approximately 20 micrometers in diameter which allowsreproducible injection of 1-2 pl of DNA solution. The use of zygotes asa target for gene transfer has a major advantage in that in most casesthe injected DNA will be incorporated into the host gene before thefirst cleavage (Brinster et al. Proc. Natl. Acad. Sci. USA 82: 4438-4442(1985)). As a consequence, all cells of the transgenic animal will carrythe incorporated transgene. This will in general also be reflected inthe efficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene.

Retroviral infection can also be used to introduce transgene into anon-human mammal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenich, R. Proc. Natl. Acad. Sci. USA73: 1260-1264 (1976)). Efficient infection of the blastomeres isobtained by enzymatic treatment to remove the zona pellucida(Manipulating the Mouse Embryo, Hogan eds., Cold Spring HarborLaboratory Press, Cold Spring Harbor, (1986)). The viral vector systemused to introduce the transgene is typically a replication-defectiveretrovirus carrying the transgene (Jahner et al. Proc. Natl. Acad. Sci.USA 82: 6927-6931 (1985)). Van der Putten et al. Proc. Natl. Acad. Sci.USA 82: 6148-6152 (1985)). Transfection is easily and efficientlyobtained by culturing the blastomeres on a monolayer of virus-producingcells (Van der Putten, supra; Stewart et al. EMBO J. 6: 383-388 (1987)).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can also be injected into the blastocoele (Jahneret al. Nature 298: 623-628 (1982)). Most of the founders will be mosaicfor the transgene since incorporation occurs only in a subset of thecells which formed the transgenic animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infection of the midgestation embryo(Jahner et al. (1982) supra).

Insertion of the transgene construct into the ES cells can beaccomplished using a variety of methods well known in the art includingfor example, electroporation, microinjection, and calcium phosphatetreatment. A preferred method of insertion is electroporation, in whichthe ES cells and the transgene construct DNA are exposed to an electricpulse using an electroporation machine and following the manufacturer'sguidelines for use. After electroporation, the ES cells are typicallyallowed to recover under suitable incubation conditions. The cells arethen screened for the presence of the transgene.

D. Implantation of Embryos

Pseudopregnant, foster or surrogate mothers are prepared for the purposeof implanting embryos, which have been modified by introducing thetransgene. Such foster mothers are typically prepared by mating withvasectomized males of the same species. The stage of the pseudopregnantfoster mother is important for successful implantation, and it isspecies dependent. For mice, this stage is about 2-3 dayspseudopregnant. Recipient females are mated at the same time as donorfemales. Although the following description relates to mice, it can beadapted for any other non-human mammal by those skilled in the art. Atthe time of embryo transfer, the recipient females are anesthetized withan intraperitoneal injection of 0.015 ml of 2.5% avertin per gram ofbody weight. The oviducts are exposed by a single midline dorsalincision. An incision is then made through the body wall directly overthe oviduct. The ovarian bursa is then torn with watchmaker's forceps.Embryos to be transferred are placed in DPBS (Dulbecco's phosphatebuffered saline) and in the tip of a transfer pipet (about 10 to 12embryos). The pipet tip is inserted into the infundibulum and theembryos transferred. After the transfer, the incision is closed by twosutures. The number of embryos implanted into a particular host willvary by species, but will usually be comparable to the number ofoffspring the species naturally produces.

Where the ES cell have been used to introduce the transgene, thetransformed ES cells are incorporated into the embryo as describedearlier, and the embryos may be implanted into the uterus of apseudopregnant foster mother for gestation.

E. Screening for the Presence or Expression of Transgene

Transgenic offspring of the surrogate host may be screened for thepresence and/or expression of the transgene by any suitable method.Offspring that are born to the foster mother may be screened initiallyfor mosaic coat color where the coat color selection strategy (asdescribed above) has been employed. Alternatively, or additionally,screening is often accomplished by Southern blot or PCR of DNA preparedfrom tail tissue, using a probe that is complementary to at least aportion of the transgene. Western blot analysis or immunohistochemistryusing an antibody against the protein encoded by the transgene may beemployed as an alternative or additional method for screening for thepresence of the transgene product. Alternatively, the tissues or cellsbelieved to express the transgene at the highest levels are tested forthe RNA expression of the transgene using Northern analysis or RT-PCR.

Alternative or additional methods for evaluating the presence of thetransgene include, without limitation, suitable biochemical assays suchas enzyme and/or immunological assays, histological stains forparticular marker or enzyme activities, flow cytometric analysis, andthe like. Analysis of the blood may also be useful to detect thepresence of the transgene product in the blood, as well as to evaluatethe effect of the transgene on the levels of various types of bloodcells and other blood constituents.

F. Breeding of the Transgenic Animals

Progeny of the transgenic animals may be obtained by mating thetransgenic animal with a suitable partner, or by in vitro fertilizationof eggs and/or sperm obtained from the transgenic animal. Where matingwith a partner is to be performed, the partner may or may not betransgenic; where it is transgenic, it may contain the same or adifferent transgene, or both. Alternatively, the partner may be aparental line. Where in vitro fertilization is used, the fertilizedembryo may be implanted into a surrogate host or incubated in vitro, orboth. Using either method, the progeny may be evaluated for the presenceof the transgene using methods described above, or other appropriatemethods. Typically, crossing and backcrossing is accomplished by matingsiblings or a parental strain with an offspring, depending on the goalof each particular step in the breeding process.

A preferred embodiment of the invention relates to mice that lack bothalleles for antibody heavy chain (IgH-KOAH^(−/−)), and have very lowlevels of circulating antibodies (Kitamura et al., Nature 350:423-426(1991)). In one aspect, the invention concerns a transgenic non-humanmammal that produces in its B-cells GIPF protein or a fragment thereof.The transgenic mammal has stably integrated into its genome a nucleicacid sequence encoding GIPF or a fragment thereof having the biologicalactivity of the native protein, operably linked to transcriptionalregulatory sequences directing its expression in B-cells. Thetranscriptional regulatory sequences preferably comprise a B-cellspecific promoter, such as the immunoglobulin kappa chain promoter.Without limitation, the non-human transgenic mammal may, for example, bemouse, rat, rabbit, pig, goat, goat or cattle.

4.2.7 Crypt Cell and Tissue Growth Activity

The GIPF polypeptide of the invention exhibits growth factor activityand is involved in the proliferation and differentiation of intestinalcrypt cells. GIPF may also exhibit growth factor activity on otherepithelial cells of the gastrointestinal tract. Administration of thepolypeptide of the invention to crypt cells in vivo or ex vivo maymaintain and expand cell populations in a totipotential state whichwould be useful for re-engineering damaged or diseased tissues,transplantation, manufacture of bio-pharmaceuticals and the developmentof bio-sensors. The ability to produce large quantities of human cellshas important working applications for the production of human proteinswhich currently must be obtained from non-human sources or donors,implantation of cells to treat tissues for grafting suchgastrointestinal cells.

It is contemplated that multiple different exogenous growth factorsand/or cytokines may be administered in combination with the polypeptideof the invention to achieve the desired effect, including any of thegrowth factors listed herein, other stem cell maintenance factors, andspecifically including stem cell factor (SCF), leukemia inhibitoryfactor (LIF), Flt-3 ligand (Flt-3L), any of the interleukins,recombinant soluble IL-6 receptorfused to IL-6, macrophage inflammatoryprotein 1-alpha (MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO),platelet factor 4 (PF-4), platelet-derived growth factor (PDGF), neuralgrowth factors, basic fibroblast growth factor (bFGF), keratinocytegrowth factor-2 (KGF2), and glucagons-like peptide 2 (GLP-2).

Intestinal epithelial cells including crypt cells can be transfectedwith a polynucleotide of the invention to induce autocrine expression ofthe polypeptide of the invention. This will allow for generation ofundifferentiated cell lines that are useful as is or that can then bedifferentiated into the desired mature cell types. These stable celllines can also serve as a source of undifferentiated mRNA to create cDNAlibraries and templates for polymerase chain reaction experiments. Thesestudies would allow for the isolation and identification ofdifferentially expressed genes in crypt cell populations that regulatecrypt proliferation and/or maintenance.

Expansion and maintenance of epithelial stem cell populations will beuseful in the treatment of many pathological conditions. For example,polypeptides of the present invention may be used to manipulate cryptcells in culture to give rise to gastrointestinal epithelial cells thatcan be used to augment or replace cells damaged by illness, autoimmunedisease, accidental damage or genetic disorders, inflammation caused byionizing radiation, chemotherapy, infection and inflammation.

Expression of the polypeptide of the invention and its effect on cryptcells can also be manipulated to achieve controlled differentiation ofthe crypt cells into more differentiated cell types. A broadlyapplicable method of obtaining pure populations of a specificdifferentiated cell type from undifferentiated stem cell populationsinvolves the use of a cell-type specific promoter driving a selectablemarker

In vitro cultures of intestinal epithelial cells including crypt cellscan be used to determine if the polypeptide of the invention exhibitsgrowth factor activity. Crypt cells are isolated from disaggregatedcolonic crypts from human and murine colonic mucosa, and the clonogenicactivity of GIPF can be assessed using the method described by Whiteheadet al., Gastroenterology 117:858-865 (1999), which is hereinincorporated by reference in its entirety. Growth factor activity may beassed in the presence of the polypeptide of the invention alone or incombination with other growth factors or cytokines.

The compositions of the present invention may also be useful forproliferation of intestinal epithelial cells including crypt cells andfor regeneration of oral and gastrointestinal tissue, i.e. for thetreatment of injuries sustained by the epithelial layer which involvedegeneration, death or trauma to epithelial crypt cells. Morespecifically, a composition may be used in the treatment of diseases ofthe gastrointestinal tract as recited herein.

Compositions of the invention may also be useful to promote better orfaster closure of non-healing wounds, including without limitationpressure ulcers, ulcers associated with vascular insufficiency, surgicaland traumatic wounds, and the like. Assays for wound healing activityinclude, without limitation, those described in: Winter, Epidermal WoundHealing, pp. 71-112 (Maibach, H. I. and Rovee, D. T., eds.), Year BookMedical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz,J. Invest. Dermatol 71:382-84 (1978).

4.2.8 Immunomodulatory Activity

A polypeptide of the present invention may exhibit activity relating toregulation of immune system components including, but not limited tocytokine production and/or activity, and/or cells of the immune system.A polynucleotide of the invention can encode a polypeptide exhibitingsuch attributes. Regulation of cytokines and/or cells of the immunesystem may include increasing and/or decreasing levels of cytokines ornumbers of particular cells of the immune system.

With such immunomodulatory activity, polypeptides of the invention maybe used to treat various immune disorders. These disorders include, butare not limited to inflammatory bowel disease (IBD), which includesulcerative colitis and/or Crohn's disease, and mucositis as aconsequence of anti-cancer therapies including radiation treatmentand/or chemotherapy. The cause of these immune disorders may be, forexample, idiopathic (i.e. of unknown cause), genetic, by infectiousagents (eg. viruses, bacteria, fungi), and/or by damage induced byanti-cancer therapies (eg. radiation therapy and/or chemotherapy).

Modulation of immune responses and/or components of the immune systemmay be accomplished in a number of ways. Down-regulation may be in theform of inhibiting or blocking an immune response already in progress ormay involve preventing the induction of an immune response. Thefunctions of activated T cells may be inhibited by suppressing T cellresponses or by inducing specific tolerance in T cells, or both.Immunosuppression of T cell responses is generally an active,non-antigen-specific, process that requires continuous exposure of the Tcells to the suppressive agent. Tolerance, which involves inducingnon-responsiveness or anergy in T cells, is distinguishable fromimmunosuppression in that it is generally antigen-specific and persistsafter exposure to the tolerizing agent has ceased. Operationally,tolerance can be demonstrated by the lack of a T cell response uponreexposure to specific antigen in the absence of tolerizing agent.

Inflammatory bowel disease is almost always mediated by one of twopathways: excessive T helper 1 (Th1)-cell response associated with highlevels of IL-12, IFN-gamma, and/or TNF or excessive T helper 2(Th2)-cell response associated with high levels of IL-4, IL-5, and/orIL-13 (Bouma et al., herein incorporated by reference in its entirety).Therefore a mechanism through which polypeptides of the invention couldmediate immunomodulatory activity in disease treatment would be todown-regulate the numbers of Th1 and/or Th2 cell populations.Alternatively, another activity could be to decrease the levels ofcytokines (eg. IL-12, IFN-gamma, TNF, IL-4, IL-5, and/or IL-13) that areassociated with and/or mediate the inflammatory response.

The activity of the polypeptide of the present invention may, amongother means, be measured by the following methods:

Assays for T-cell or thymocyte proliferation include without limitationthose described in: Current Protocols in Immunology, Ed by J. E.Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, InVitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7,Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolliet al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., I.Immunol. 149:3778-3783, 1992; Bowman et al., I. Immunol. 152:1756-1761,1994.

Assays for cytokine production and/or proliferation of spleen cells,lymph node cells or thymocytes include, without limitation, thosedescribed in: Polyclonal T cell stimulation, Kruisbeek, A. M. andShevach, E. M. In Current Protocols in Immunology. J. E. e.a. Coliganeds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; andMeasurement of mouse and human interferon-γ, Schreiber, R. D. In CurrentProtocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8,John Wiley and Sons, Toronto. 1994.

Assays for T-cell clone responses to antigens (which will identify,among others, proteins that affect APC-T cell interactions as well asdirect T-cell effects by measuring proliferation and cytokineproduction) include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction; Chapter 6, Cytokines and their cellular receptors; Chapter 7,Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad.Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun.11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takaiet al., J. Immunol. 140:508-512, 1988.)

4.2.9 Chemotactic/Chemokinetic Activity

A polypeptide of the present invention may be involved in chemotactic orchemokinetic activity for mammalian cells, including, for example,monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils,epithelial and/or endothelial cells. A polynucleotide of the inventioncan encode a polypeptide exhibiting such attributes. Chemotactic andchemokinetic receptor activation can be used to mobilize or attract adesired cell population to a desired site of action. Chemotactic orchemokinetic compositions (e.g. proteins, antibodies, binding partners,or modulators of the invention) provide particular advantages intreatment of wounds and other trauma to tissues, as well as in treatmentof localized infections. For example, attraction of lymphocytes,monocytes or neutrophils to tumors or sites of infection may result inimproved immune responses against a tumor or an infecting agent.

A protein or peptide has chemotactic activity for a particular cellpopulation if it can stimulate, directly or indirectly, the directedorientation or movement of such cell population. Preferably, the proteinor peptide has the ability to directly stimulate directed movement ofcells. Whether a particular protein has chemotactic activity for apopulation of cells can be readily determined by employing such proteinor peptide in any known assay for cell chemotaxis.

Assays for chemotactic activity (which will identify proteins thatinduce or prevent chemotaxis) consist of assays that measure the abilityof a protein to induce the migration of cells across a membrane as wellas the ability of a protein to induce the adhesion of one cellpopulation to another cell population. Suitable assays for movement andadhesion include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Marguiles, E. M. Shevach, W. Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 6.12, Measurement of alpha and betaChemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376,1995; Lind et al. APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol.25:1744-1748; Gruberetal. J. of Immunol. 152:5860-5867, 1994; Johnstonet al. J. of Immunol. 153:1762-1768, 1994.

4.2.10 Drug Screening

The transgenic non-human mammals and their progeny of the presentinvention provide several important uses that will be readily apparentto one of ordinary skill in the art. The transgenic animals areparticularly useful in screening compounds that modulate (i.e. increaseor decrease) the activity of the GIPF polypeptides. Screening for auseful compound involves administering the candidate compound over arange of doses to the transgenic animal, and assaying at various timepoints for the effect(s) of the compound on the activity of the GIPFprotein. The compound may be administered prior to or at the onset ofabdominal distension. Administration may be oral, or by suitableinjection, depending on the chemical nature of the compound beingevaluated. The cellular response to the compound is evaluated over timeusing appropriate biochemical and/or histological assays.

Sources for test compounds that may be screened for ability to bind toor modulate (i.e., increase or decrease) the activity of polypeptides ofthe invention include (1) inorganic and organic chemical libraries, (2)natural product libraries, and (3) combinatorial libraries comprised ofeither random or mimetic peptides, oligonucleotides or organicmolecules.

Chemical libraries may be readily synthesized or purchased from a numberof commercial sources, and may include structural analogs of knowncompounds or compounds that are identified as “hits” or “leads” vianatural product screening.

The sources of natural product libraries are microorganisms (includingbacteria and fungi), animals, plants or other vegetation, or marineorganisms, and libraries of mixtures for screening may be created by:(1) fermentation and extraction of broths from soil, plant or marinemicroorganisms or (2) extraction of the organisms themselves. Naturalproduct libraries include polyketides, non-ribosomal peptides, and(non-naturally occurring) variants thereof. For a review, see Science282:63-68 (1998).

Combinatorial libraries are composed of large numbers of peptides,oligonucleotides or organic compounds and can be readily prepared bytraditional automated synthesis methods, PCR, cloning or proprietarysynthetic methods. Of particular interest are peptide andoligonucleotide combinatorial libraries. Still other libraries ofinterest include peptide, protein, peptidomimetic, multiparallelsynthetic collection, recombinatorial, and polypeptide libraries. For areview of combinatorial chemistry and libraries created therefrom, seeMyers, Curr. Opin. Biotechnol. 8:701-707 (1997). For reviews andexamples of peptidomimetic libraries, see Al-Obeidi et al., Mol.Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol,1(1):114-19 (1997); Dorner et al., Bioorg Med Chem, 4(5):709-15 (1996)(alkylated dipeptides).

4.3 Diseases Amenable to GIPF Therapy

In one aspect, the present invention provides pharmaceutical reagentsand methods useful for treating diseases and conditions whereinepithelialization is desired. GIPF polypeptides are useful to increasecytoprotection, proliferation and/or differentiation of epithelial cellsof the oral and gastrointestinal tract. Specifically, GIPF polypeptidesare useful to treat or prevent diseases or conditions that includewithout limitation gastrointestinal diseases, mucositis of thegastrointestinal tract, mucositis of the oropharynx, lips and esophagus(oral mucositis), inflammatory bowel disease, short bowel syndrome,gastric and duodenal ulcers, erosions of the gastrointestinal tractincluding erosive gastritis, esophagitis, esophageal reflux and otherconditions including wounds, burns, ophthalmic disorders, and anydisorder where stimulation of epithelial cell proliferation orregeneration is desired. Treatment of diseases that result ininsufficient production of mucus throughout the oral andgastrointestinal tract is also contemplated.

Mucositis, which includes oral and gastrointestinal mucositis, is acomplication of some cancer therapies in which the lining of thedigestive system becomes inflamed. GIPF is useful for preventing and/orameliorating the degeneration of the mucosa of the alimentary tract thatis caused by chemotherapy and/or radiation therapy given to a patientfor the treatment of cancer, or is given as an adjuvant therapyfollowing the removal of a tumor. Exemplary chemotherapeutic agentsinclude, without limitation, BCNU, busulfan, carboplatin,cyclophosphamide, tannorubicin, doxorubicin, etoposide, 5-fluorouracil,gemcitabine, ifophamide, irinotecan, melphalan, methotrexate, navelbine,topotecan, and taxol, and exemplary treatment regimens include withoutlimitation, BEAM (busulfan, etoposide, cytosine, arabinoside,methotrexate); cyclophosphamide and total body irradiation;cyclophosphamine, total body irradiation and etoposide; cyclophosphamideand busulfan; and 5-fluorouracil with leucovorin or levamisole.Treatment, pretreatment or post-treatment with GIPF is useful togenerate a cytoprotective effect or regeneration or both, for example,of the mucosa of the small intestine and colon, allowing increaseddosages of therapies while reducing their potential side effects.

Inflammatory bowel disease that can be treated with GIPF includesgeneral inflammatory bowel disease that is characterized by chronic,relapsing, inflammatory disorders of unknown origin, Crohn's disease,dysplasia associated with inflammatory bowel disease, intermediatecolitis, ulcerative colitis; non-infectious colitis including activecolitis, antibiotic-associated colitis, collagenous colitis, diversioncolitis, eosinophilic colitis, graft versus host disease, granulomatouscolitis, ischaemic colitis, hemorrhagic colitis, malacoplakia,necrotizing enterocolitis, radiation enterocolitis, typhlitis;infectious colitis including adenovirus and amebic colitis,balantidiasis, HSV/AIDS associated colitis, and colitis caused bytrypanosomes, E. coli, Mycobacterium avium intracellulare, Sotavirus,Salmonella, Shigella, Campylobacter jejuni, Clostridium, Botulinum, andcolitis associated with schistosomiasis, spirochetosis, syphilis,trichuriasis, tuberculosis typhoid fever, Vibrio cholera, and Yersinia.

Short bowel syndrome is a group of problems affecting people who havehad half or more of their small intestine removed. The most commonreason for removing part of the small intestine is to treat Crohn'sdisease. In addition, surgical resection of part of the intestine may berequired to remove cancerous growths. Diarrhea is the main symptom ofshort bowel syndrome. Other symptoms include cramping, bloating, andheartburn. Many people with short bowel syndrome are malnourishedbecause their remaining small intestine is unable to absorb enoughwater, vitamins, and other nutrients from food. They may also becomedehydrated, which can be life threatening. Problems associated withdehydration and malnutrition include weakness, fatigue, depression,weight loss, bacterial infections, and food sensitivities. Short bowelsyndrome is treated through changes in diet, intravenous feeding,vitamin and mineral supplements, and medicine to relieve symptoms. GIPFpolypeptides may be useful to increase the proliferation of theunresected intestinal tissue, thereby increasing the absorptive surfacearea of the intestine, and ameliorate the symptoms associated with shortbowel syndrome.

The cytoprotective and/or regenerative activity of GIPF polypeptides canbe tested in in vivo models of radiation induced mucositis (Withers andElkind, Int J Radiat 17:261-267 (1970), herein incorporated byreference) in in vivo chemotherapy-induced mucositis (Soris et al., OralSurg Oral Med Oral Pathol 69:437-443 (1990); Moore, Cancer ChemotherPharmacol 15:11-15 (1985); Farell et al., Cell Prolif 35:78-85 (2002),all of which are incorporated by reference in their entirety); in adextran sulfate sodium (DSS) model of colitis and small intestinalulceration or inflammation (Jeffers et al., Gastroenterology123:1151-1162 (2002), Han et al., Am J Physiol Gastrointest LiverPhysiol 279:G1011-G1022 (2000); and in a surgical model of short bowelsyndrome (SBS) (Scott et al. Am J Physiol G911-G921 (1998); Helmrath etal., J Am Coll Surg 183:441-449 (1996)), herein incorporated byreference in their entirety).

Comparisons of GIPF mRNA and protein expression levels between diseasedcells, tissue and corresponding normal samples are made to determine ifthe subject is responsive to GIPF therapy. Methods for detecting andquantifying the expression of GIPF polypeptide mRNA or protein usestandard nucleic acid and protein detection and quantitation techniquesthat are well known in the art and are described in Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,NY (1989) or Ausubel, et al., Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y. (1989), both of which are incorporatedherein by reference in their entirety. Standard methods for thedetection and quantification of GIPF mRNA include in situ hybridizationusing labeled GIPF riboprobes (Gemou-Engesaeth, et al., Pediatrics 109:E24-E32 (2002), herein incorporated by reference in its entirety),Northern blot and related techniques using GIPF polynucleotide probes(Kunzli, et al., Cancer 94: 228 (2002), herein incorporated by referencein its entirety, herein incorporated by reference in its entirety),RT-PCR analysis using GIPF-specific primers (Angchaiskisiri, et al.,Blood 99:130 (2002)), and other amplification detection methods, such asbranched chain DNA solution hybridization assay (Jardi, et al., J. ViralHepat. 8:465-471 (2001), herein incorporated by reference in itsentirety), transcription-mediated amplification (Kimura, et al., J.Clin. Microbiol. 40:439-445 (2002)), microarray products, such asoligos, cDNAs, and monoclonal antibodies, and real-time PCR (Simpson, etal., Molec. Vision, 6:178-183 (2000), herein incorporated by referencein its entirety). Standard methods for the detection and quantificationof GIPF protein include western blot analysis (Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,NY (1989), Ausubel, et al., Current Protocols in Molecular Biology, JohnWiley & Sons, New York, N.Y. (1989)), immunocytochemistry (Racila, etal., Proc. Natl. Acad. Sci. USA 95:4589-4594 (1998) supra), and avariety of immunoassays, including enzyme-linked immunosorbant assay(ELISA), radioimmuno assay (RIA), and specific enzyme immunoassay (EIA)(Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, NY (1989), Ausubel, et al., Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y. (1989)).

The diseases and conditions treatable by methods of the presentinvention preferably occur in mammals. Mammals include, for example,humans and other primates, as well as pet or companion animals such asdogs and cats, laboratory animals such as rats, mice and rabbits, andfarm animals such as horses, pigs, sheep, and cattle.

4.3.1 Therapeutic Methods

The compositions (including polypeptide fragments, analogs, variants andantibodies or other binding partners or modulators including antisensepolynucleotides) of the invention have numerous applications in avariety of therapeutic methods. Examples of therapeutic applicationsinclude, but are not limited to, those exemplified herein.

One embodiment of the invention is the administration of an effectiveamount of GIPF polypeptides or other composition of the invention toindividuals affected by a disease or disorder that can be treated thepeptides of the invention. While the mode of administration is notparticularly important, parenteral administration is preferred.Exemplary modes of administration are to deliver a subcutaneous orintravenous bolus. The dosage of GIPF polypeptides or other compositionof the invention will normally be determined by the prescribingphysician. It is to be expected that the dosage will vary according tothe age, weight, condition and response of the individual patient.Typically, the amount of polypeptide administered per dose will be inthe range of about 0.01 μg/kg to 100 mg/kg of body weight, with thepreferred dose being about 0.1 μg/kg to 10 mg/kg of patient body weight.For parenteral administration, GIPF polypeptides of the invention willbe formulated in an injectable form combined with a pharmaceuticallyacceptable parenteral vehicle. Such vehicles are well known in the artand examples include water, saline, Ringer's solution, dextrosesolution, and solutions consisting of small amounts of the human serumalbumin. The vehicle may contain minor amounts of additives thatmaintain the isotonicity and stability of the polypeptide or otheractive ingredient. The preparation of such solutions is within the skillof the art.

4.3.2 Pharmaceutical Formulations

A protein or other composition of the present invention (from whateversource derived, including without limitation from recombinant andnon-recombinant sources and including antibodies and other bindingpartners of the polypeptides of the invention) may be administered to apatient in need, by itself, or in pharmaceutical compositions where itis mixed with suitable carriers or excipient(s) at doses to treat orameliorate a variety of disorders. Such a composition may optionallycontain (in addition to protein or other active ingredient and acarrier) diluents, fillers, salts, buffers, stabilizers, solubilizers,and other materials well known in the art. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).The characteristics of the carrier will depend on the route ofadministration. The pharmaceutical composition of the invention may alsocontain cytokines, lymphokines, or other hematopoietic factors andvarious growth factors such as any of the FGFs, epidermal growth factor(EGF), platelet-derived growth factor (PDGF), transforming growthfactors (TGF-α and TGF-β), insulin-like growth factor (IGF),keratinocyte growth factor (KGF), and the like, as well as cytokinesdescribed herein.

The pharmaceutical composition may further contain other agents whicheither enhance the activity of the protein or other active ingredient orcomplement its activity or use in treatment. Such additional factorsand/or agents may be included in the pharmaceutical composition toproduce a synergistic effect with protein or other active ingredient ofthe invention, or to minimize side effects. Conversely, protein or otheractive ingredients of the present invention may be included informulations of the particular cytokine, lymphokine, other hematopoieticfactor, thrombolytic or anti-thrombotic factor, or anti-inflammatoryagent to minimize side effects of the clotting factor, cytokine,lymphokine, other hematopoietic factor, thrombolytic or anti-thromboticfactor, or anti-inflammatory agent (such as IL-1 Ra, IL-1 Hy1, IL-1 Hy2,anti-TNF, corticosteroids, immunosuppressive agents). A protein of thepresent invention may be active in multimers (e.g., heterodimers orhomodimers) or complexes with itself or other proteins. As a result,pharmaceutical compositions of the invention may comprise a protein ofthe invention in such multimeric or complexed form.

As an alternative to being included in a pharmaceutical composition ofthe invention including a first protein, a second protein or atherapeutic agent may be concurrently administered with the firstprotein (e.g., at the same time, or at differing times provided thattherapeutic concentrations of the combination of agents is achieved atthe treatment site). Techniques for formulation and administration ofthe compounds of the instant application may be found in “Remington'sPharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latestedition. A therapeutically effective dose further refers to that amountof the compound sufficient to result in amelioration of symptoms, e.g.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, a therapeutically effective dose refersto that ingredient alone. When applied to a combination, atherapeutically effective dose refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredin combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of protein or other active ingredientof the present invention is administered to a mammal having a conditionto be treated. Protein or other active ingredient of the presentinvention may be administered in accordance with the method of theinvention either alone or in combination with other therapies such astreatments employing cytokines, lymphokines or other hematopoieticfactors. When co-administered with one or more cytokines, lymphokines orother hematopoietic factors, protein or other active ingredient of thepresent invention may be administered either simultaneously with thecytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolyticor anti-thrombotic factors, or sequentially. If administeredsequentially, the attending physician will decide on the appropriatesequence of administering protein or other active ingredient of thepresent invention in combination with cytokine(s), lymphokine(s), otherhematopoietic factor(s), thrombolytic or anti-thrombotic factors.

4.3.3 Routes of Administration

Suitable routes of administration may, for example, include oral,rectal, transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections. Administrationof protein or other active ingredient of the present invention used inthe pharmaceutical composition or to practice the method of the presentinvention can be carried out in a variety of conventional ways, such asoral ingestion, inhalation, topical application or cutaneous,subcutaneous, intraperitoneal (IP), parenteral or intravenous injection.Intravenous administration to the patient is preferred.

Alternatively, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto the tissue, often in a depot or sustained release formulation.

In another embodiment, the implantation of cells producing GIPF (celltherapy) into a subject in need of proliferation and/or stimulation ofepithelial cells is contemplated. Cells that do not normally expressGIPF or that express low levels of GIPF may be modified to producetherapeutic levels of GIPF by transformation with a polynucleotide thatencodes GIPF. The cells may be of the same species as the subject, ormay be derived from a different species. Preferably, the cells arederived from the subject in need of GIPF therapy. Human or nonhumancells may be implanted in a subject using a biocompatible,semi-permeable polymeric enclosure to allow release of GIPF protein, ormay be implanted directly without encapsulation.

In another embodiment, in vivo gene therapy is contemplated. Anucleotide sequence encoding GIPF is introduced directly into a subjectfor secretion of the protein to prevent or treat the diseases as recitedherein. The nucleotide encoding GIPF may be injected directly into thetissue to be treated, or it may be delivered into the cells of theaffected tissue by a viral vector e.g. adenovirus vector or retrovirusvector. Physical transfer of appropriate vectors containing aGIPF-encoding nucleic acid may also be achieved by methods includingliposome-mediated transfer, direct injection of naked DNA,receptor-mediated transfer, or microparticle bombardment.

The polypeptides of the invention are administered by any route thatdelivers an effective dosage to the desired site of action. Thedetermination of a suitable route of administration and an effectivedosage for a particular indication is within the level of skill in theart. Preferably for wound treatment, one administers the therapeuticcompound directly to the site. Suitable dosage ranges for thepolypeptides of the invention can be extrapolated from these dosages orfrom similar studies in appropriate animal models. Dosages can then beadjusted as necessary by the clinician to provide maximal therapeuticbenefit.

4.3.4 Compositions/Formulations

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in a conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. These pharmaceuticalcompositions may be manufactured in a manner that is itself known, e.g.,by means of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Proper formulation is dependent upon the route ofadministration chosen. When a therapeutically effective amount ofprotein or other active ingredient of the present invention isadministered orally, protein or other active ingredient of the presentinvention will be in the form of a tablet, capsule, powder, solution orelixir. When administered in tablet form, the pharmaceutical compositionof the invention may additionally contain a solid carrier such as agelatin or an adjuvant. The tablet, capsule, and powder contain fromabout 5 to 95% protein or other active ingredient of the presentinvention, and preferably from about 25 to 90% protein or other activeingredient of the present invention. When administered in liquid form, aliquid carrier such as water, petroleum, oils of animal or plant originsuch as peanut oil, mineral oil, soybean oil, or sesame oil, orsynthetic oils may be added. The liquid form of the pharmaceuticalcomposition may further contain physiological saline solution, dextroseor other saccharide solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol. When administered in liquidform, the pharmaceutical composition contains from about 0.5 to 90% byweight of protein or other active ingredient of the present invention,and preferably from about 1 to 50% protein or other active ingredient ofthe present invention.

When a therapeutically effective amount of protein or other activeingredient of the present invention is administered by intravenous,cutaneous or subcutaneous injection, protein or other active ingredientof the present invention will be in the form of a pyrogen-free,parenterally acceptable aqueous solution. The preparation of suchparenterally acceptable protein or other active ingredient solutions,having due regard to pH, isotonicity, stability, and the like, is withinthe skill in the art. A preferred pharmaceutical composition forintravenous, cutaneous, or subcutaneous injection should contain, inaddition to protein or other active ingredient of the present invention,an isotonic vehicle such as Sodium Chloride Injection, Ringer'sInjection, Dextrose Injection, Dextrose and Sodium Chloride Injection,Lactated Ringer's Injection, or other vehicle as known in the art. Thepharmaceutical composition of the present invention may also containstabilizers, preservatives, buffers, antioxidants, or other additivesknown to those of skill in the art. For injection, the agents of theinvention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiological saline buffer. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration. For buccal administration, the compositions may take theform of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch. The compounds maybe formulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides. In additionto the formulations described previously, the compounds may also beformulated as a depot preparation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

A pharmaceutical carrier for the hydrophobic compounds of the inventionis a co-solvent system comprising benzyl alcohol, a nonpolar surfactant,a water-miscible organic polymer, and an aqueous phase. The co-solventsystem may be the VPD co-solvent system. VPD is a solution of 3% w/vbenzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5%dextrose in water solution. This co-solvent system dissolves hydrophobiccompounds well, and itself produces low toxicity upon systemicadministration. Naturally, the proportions of a co-solvent system may bevaried considerably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied: for example, other low-toxicity nonpolar surfactants maybe used instead of polysorbate 80; the fraction size of polyethyleneglycol may be varied; other biocompatible polymers may replacepolyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars orpolysaccharides may substitute for dextrose. Alternatively, otherdelivery systems for hydrophobic pharmaceutical compounds may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs. Certain organic solventssuch as dimethylsulfoxide also may be employed, although usually at thecost of greater toxicity. Additionally, the compounds may be deliveredusing a sustained-release system, such as semipermeable matrices ofsolid hydrophobic polymers containing the therapeutic agent. Varioustypes of sustained-release materials have been established and are wellknown by those skilled in the art. Sustained-release capsules may,depending on their chemical nature, release the compounds for a fewweeks up to over 100 days. Depending on the chemical nature and thebiological stability of the therapeutic reagent, additional strategiesfor protein or other active ingredient stabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols. Many of the active ingredients of theinvention may be provided as salts with pharmaceutically compatiblecounter ions. Such pharmaceutically acceptable base addition salts arethose salts which retain the biological effectiveness and properties ofthe free acids and which are obtained by reaction with inorganic ororganic bases such as sodium hydroxide, magnesium hydroxide, ammonia,trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodiumacetate, potassium benzoate, triethanol amine and the like.

The pharmaceutical composition of the invention may be in the form of acomplex of the protein(s) or other active ingredient of presentinvention along with protein or peptide antigens. The protein and/orpeptide antigen will deliver a stimulatory signal to both B and Tlymphocytes. B lymphocytes will respond to antigen through their surfaceimmunoglobulin receptor. T lymphocytes will respond to antigen throughthe T cell receptor (TCR) following presentation of the antigen by MHCproteins. MHC and structurally related proteins including those encodedby class I and class II MHC genes on host cells will serve to presentthe peptide antigen(s) to T lymphocytes. The antigen components couldalso be supplied as purified MHC-peptide complexes alone or withco-stimulatory molecules that can directly signal T cells. Alternativelyantibodies able to bind surface immunoglobulin and other molecules on Bcells as well as antibodies able to bind the TCR and other molecules onT cells can be combined with the pharmaceutical composition of theinvention.

The pharmaceutical composition of the invention may be in the form of aliposome in which protein of the present invention is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids which exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers in aqueoussolution. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithins,phospholipids, saponin, bile acids, and the like. Preparation of suchliposomal formulations is within the level of skill in the art, asdisclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728;4,837,028; and 4,737,323, all of which are incorporated herein byreference.

The amount of protein or other active ingredient of the presentinvention in the pharmaceutical composition of the present inventionwill depend upon the nature and severity of the condition being treated,and on the nature of prior treatments which the patient has undergone.Ultimately, the attending physician will decide the amount of protein orother active ingredient of the present invention with which to treateach individual patient. Initially, the attending physician willadminister low doses of protein or other active ingredient of thepresent invention and observe the patient's response. Larger doses ofprotein or other active ingredient of the present invention may beadministered until the optimal therapeutic effect is obtained for thepatient, and at that point the dosage is not increased further. It iscontemplated that the various pharmaceutical compositions used topractice the method of the present invention should contain about 0.01μg to about 100 mg (preferably about 0.1 μg to about 10 mg, morepreferably about 0.1 μg to about 1 mg) of protein or other activeingredient of the present invention per kg body weight. For compositionsof the present invention which are useful for bone, cartilage, tendon orligament regeneration, the therapeutic method includes administering thecomposition topically, systematically, or locally as an implant ordevice. When administered, the therapeutic composition for use in thisinvention is, of course, in a pyrogen-free, physiologically acceptableform. Further, the composition may desirably be encapsulated or injectedin a viscous form for delivery to the site of bone, cartilage or tissuedamage. Topical administration may be suitable for wound healing andtissue repair. Therapeutically useful agents other than a protein orother active ingredient of the invention which may also optionally beincluded in the composition as described above, may alternatively oradditionally, be administered simultaneously or sequentially with thecomposition in the methods of the invention. Preferably for bone and/orcartilage formation, the composition would include a matrix capable ofdelivering the protein-containing or other active ingredient-containingcomposition to the site of bone and/or cartilage damage, providing astructure for the developing bone and cartilage and optimally capable ofbeing resorbed into the body. Such matrices may be formed of materialspresently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined calciumsulfate, tricalcium phosphate, hydroxyapatite, polylactic acid,polyglycolic acid and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sinteredhydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may becomprised of combinations of any of the above mentioned types ofmaterial, such as polylactic acid and hydroxyapatite or collagen andtricalcium phosphate. The bioceramics may be altered in composition,such as in calcium-aluminate-phosphate and processing to alter poresize, particle size, particle shape, and biodegradability. Presentlypreferred is a 50:50 (mole weight) copolymer of lactic acid and glycolicacid in the form of porous particles having diameters ranging from 150to 800 microns. In some applications, it will be useful to utilize asequestering agent, such as carboxymethyl cellulose or autologous bloodclot, to prevent the protein compositions from disassociating from thematrix.

A preferred family of sequestering agents is cellulosic materials suchas alkylcelluloses (including hydroxyalkylcelluloses), includingmethylcellulose, ethylcellulose, hydroxyethyl cellulose,hydroxypropylcellulose, hydroxypropyl-methylcellulose, andcarboxymethylcellulose, the most preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate, poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). Theamount of sequestering agent useful herein is 0.5-20 wt %, preferably1-10 wt % based on total formulation weight, which represents the amountnecessary to prevent desorption of the protein from the polymer matrixand to provide appropriate handling of the composition, yet not so muchthat the progenitor cells are prevented from infiltrating the matrix,thereby providing the protein the opportunity to assist the osteogenicactivity of the progenitor cells. In further compositions, proteins orother active ingredient of the invention may be combined with otheragents beneficial to the treatment of the bone and/or cartilage defect,wound, or tissue in question. These agents include various growthfactors such as epidermal growth factor (EGF), platelet derived growthfactor (PDGF), transforming growth factors (TGF-α and TGF-β), andinsulin-like growth factor (IGF).

The therapeutic compositions are also presently valuable for veterinaryapplications. Particularly domestic animals and thoroughbred horses, inaddition to humans, are desired patients for such treatment withproteins or other active ingredient of the present invention. The dosageregimen of a protein-containing pharmaceutical composition to be used intissue regeneration will be determined by the attending physicianconsidering various factors which modify the action of the proteins,e.g., amount of tissue weight desired to be formed, the site of damage,the condition of the damaged tissue, the size of a wound, type ofdamaged tissue (e.g., bone), the patient's age, sex, and diet, theseverity of any infection, time of administration and other clinicalfactors. The dosage may vary with the type of matrix used in thereconstitution and with inclusion of other proteins in thepharmaceutical composition. For example, the addition of other knowngrowth factors, such as IGF I (insulin like growth factor 1), to thefinal composition, may also effect the dosage. Progress can be monitoredby periodic assessment of tissue/bone growth and/or repair, for example,X-rays, histomorphometric determinations and tetracycline labeling.

Polynucleotides of the present invention can also be used for genetherapy. Such polynucleotides can be introduced either in vivo or exvivo into cells for expression in a mammalian subject. Polynucleotidesof the invention may also be administered by other known methods forintroduction of nucleic acid into a cell or organism (including, withoutlimitation, in the form of viral vectors or naked DNA). Cells may alsobe cultured ex vivo in the presence of proteins of the present inventionin order to proliferate or to produce a desired effect on or activity insuch cells. Treated cells can then be introduced in vivo for therapeuticpurposes.

4.3.5 Effective Dosage

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. More specifically, atherapeutically effective amount means an amount effective to preventdevelopment of or to alleviate the existing symptoms of the subjectbeing treated. Determination of the effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein. For any compound used in the methodof the invention, the therapeutically effective dose can be estimatedinitially from appropriate in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat can be used to more accurately determine useful doses in humans.For example, a dose can be formulated in animal models to achieve acirculating concentration range that includes the IC₅₀ as determined incell culture (i.e., the concentration of the test compound whichachieves a half-maximal inhibition of the protein's biologicalactivity). Such information can be used to more accurately determineuseful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms or a prolongation of survivalin a patient. Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratiobetween LD₅₀ and ED₅₀. Compounds which exhibit high therapeutic indicesare preferred. The data obtained from these cell culture assays andanimal studies can be used in formulating a range of dosage for use inhuman. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. See, e.g.,Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1 p. 1. Dosage amount and interval may be adjusted individually toprovide plasma levels of the active moiety which are sufficient tomaintain the desired effects, or minimal effective concentration (MEC).The MEC will vary for each compound but can be estimated from in vitrodata. Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. However, HPLC assays orbioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen which maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%. In cases of local administration or selectiveuptake, the effective local concentration of the drug may not be relatedto plasma concentration.

An exemplary dosage regimen for polypeptides or other compositions ofthe invention will be in the range of about 0.01 μg/kg to 100 mg/kg ofbody weight daily, with the preferred dose being about 0.1 μg/kg to 25mg/kg of patient body weight daily, varying in adults and children.Dosing may be once daily, or equivalent doses may be delivered at longeror shorter intervals.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's age and weight, the severityof the affliction, the manner of administration and the judgment of theprescribing physician.

4.3.6 Diagnostic Assays And Kits

The present invention further provides methods to identify the presenceor expression of one of the ORFs of the present invention, or homologthereof, in a test sample, using a nucleic acid probe or antibodies ofthe present invention, optionally conjugated or otherwise associatedwith a suitable label.

In general, methods for detecting a polynucleotide of the invention cancomprise contacting a sample with a compound that binds to and forms acomplex with the polynucleotide for a period sufficient to form thecomplex, and detecting the complex, so that if a complex is detected, apolynucleotide of the invention is detected in the sample. Such methodscan also comprise contacting a sample under stringent hybridizationconditions with nucleic acid primers that anneal to a polynucleotide ofthe invention under such conditions, and amplifying annealedpolynucleotides, so that if a polynucleotide is amplified, apolynucleotide of the invention is detected in the sample.

In general, methods for detecting a polypeptide of the invention cancomprise contacting a sample with a compound that binds to and forms acomplex with the polypeptide for a period sufficient to form thecomplex, and detecting the complex, so that if a complex is detected, apolypeptide of the invention is detected in the sample.

In detail, such methods comprise incubating a test sample with one ormore of the antibodies or one or more of the nucleic acid probes of thepresent invention and assaying for binding of the nucleic acid probes orantibodies to components within the test sample.

Conditions for incubating a nucleic acid probe or antibody with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid probe or antibody used in the assay. One skilled in the artwill recognize that any one of the commonly available hybridization,amplification or immunological assay formats can readily be adapted toemploy the nucleic acid probes or antibodies of the present invention.Examples of such assays can be found in Chard, T., An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985). The test samplesof the present invention include cells, protein or membrane extracts ofcells, or biological fluids such as sputum, blood, serum, plasma, orurine. The test sample used in the above-described method will varybased on the assay format, nature of the detection method and thetissues, cells or extracts used as the sample to be assayed. Methods forpreparing protein extracts or membrane extracts of cells are well knownin the art and can be readily be adapted in order to obtain a samplewhich is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention. Specifically, the invention provides a compartment kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the probes or antibodies of thepresent invention; and (b) one or more other containers comprising oneor more of the following: wash reagents, reagents capable of detectingpresence of a bound probe or antibody.

In detail, a compartment kit includes any kit in which reagents arecontained in separate containers. Such containers include small glasscontainers, plastic containers or strips of plastic or paper. Suchcontainers allows one to efficiently transfer reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated, and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother. Such containers will include a container which will accept thetest sample, a container which contains the antibodies used in theassay, containers which contain wash reagents (such as phosphatebuffered saline, Tris-buffers, etc.), and containers which contain thereagents used to detect the bound antibody or probe. Types of detectionreagents include labeled nucleic acid probes, labeled secondaryantibodies, or in the alternative, if the primary antibody is labeled,the enzymatic, or antibody binding reagents which are capable ofreacting with the labeled antibody. One skilled in the art will readilyrecognize that the disclosed probes and antibodies of the presentinvention can be readily incorporated into one of the established kitformats which are well known in the art.

4.3.7 Screening Assays

Using the isolated proteins and polynucleotides of the invention, thepresent invention further provides methods of obtaining and identifyingmodulatory agents which bind to a polypeptide encoded by an ORFcorresponding to the nucleotide sequence set forth in SEQ ID NO: 2, 3,5, 9, 11, 13, 15, 17, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104 or177, or bind to a specific domain of the polypeptide encoded by thenucleic acid. In detail, said method comprises the steps of:

(a) contacting an agent with an isolated protein encoded by an ORF ofthe present invention, or nucleic acid of the invention; and

(b) determining whether the agent binds to said protein or said nucleicacid.

The modulatory agents may increase or decrease the proliferativeactivity of GIPF on epithelial cells.

In general, such methods for identifying compounds that bind to apolynucleotide of the invention can comprise contacting a compound witha polynucleotide of the invention for a time sufficient to form apolynucleotide/compound complex, and detecting the complex, so that if apolynucleotide/compound complex is detected, a compound that binds to apolynucleotide of the invention is identified.

Likewise, in general, therefore, such methods for identifying compoundsthat bind to a polypeptide of the invention can comprise contacting acompound with a polypeptide of the invention for a time sufficient toform a polypeptide/compound complex, and detecting the complex, so thatif a polypeptide/compound complex is detected, a compound that binds toa polynucleotide of the invention is identified.

Methods for identifying compounds that bind to a polypeptide of theinvention can also comprise contacting a compound with a polypeptide ofthe invention in a cell for a time sufficient to form apolypeptide/compound complex, wherein the complex drives expression of atarget gene sequence in the cell, and detecting the complex by detectingreporter gene sequence expression, so that if a polypeptide/compoundcomplex is detected, a compound that binds a polypeptide of theinvention is identified.

Compounds identified via such methods can include compounds whichmodulate the activity of a polypeptide of the invention (that is,increase or decrease its activity, relative to activity observed in theabsence of the compound). Alternatively, compounds identified via suchmethods can include compounds which modulate the expression of apolynucleotide of the invention (that is, increase or decreaseexpression relative to expression levels observed in the absence of thecompound). Compounds, such as compounds identified via the methods ofthe invention, can be tested using standard assays well known to thoseof skill in the art for their ability to modulate activity/expression.

The agents screened in the above assay can be, but are not limited to,peptides, carbohydrates, vitamin derivatives, or other pharmaceuticalagents. The agents can be selected and screened at random or rationallyselected or designed using protein modeling techniques.

For random screening, agents such as peptides, carbohydrates,pharmaceutical agents and the like are selected at random and areassayed for their ability to bind to the protein encoded by the ORF ofthe present invention. Alternatively, agents may be rationally selectedor designed. As used herein, an agent is said to be “rationally selectedor designed” when the agent is chosen based on the configuration of theparticular protein. For example, one skilled in the art can readilyadapt currently available procedures to generate peptides,pharmaceutical agents and the like, capable of binding to a specificpeptide sequence, in order to generate rationally designed antipeptidepeptides, for example see Hurby et al., Application of SyntheticPeptides: Antisense Peptides,” In Synthetic Peptides, A User's Guide,W.H. Freeman, NY (1992), pp. 289-307, and Kaspczak et al., Biochemistry28:9230-8 (1989), or pharmaceutical agents, or the like.

In addition to the foregoing, one class of agents of the presentinvention, as broadly described, can be used to control gene expressionthrough binding to one of the ORFs or EMFs of the present invention. Asdescribed above, such agents can be randomly screened or rationallydesigned/selected. Targeting the ORF or EMF allows a skilled artisan todesign sequence specific or element specific agents, modulating theexpression of either a single ORF or multiple ORFs which rely on thesame EMF for expression control. One class of DNA binding agents areagents which contain base residues which hybridize or form a triplehelix formation by binding to DNA or RNA. Such agents can be based onthe classic phosphodiester, ribonucleic acid backbone, or can be avariety of sulfhydryl or polymeric derivatives which have baseattachment capacity.

Agents suitable for use in these methods usually contain 20 to 40 basesand are designed to be complementary to a region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triplehelix-formation optimally results in a shut-off of RNA transcriptionfrom DNA, while antisense RNA hybridization blocks translation of anmRNA molecule into polypeptide. Both techniques have been demonstratedto be effective in model systems. Information contained in the sequencesof the present invention is necessary for the design of an antisense ortriple helix oligonucleotide and other DNA binding agents.

Agents which bind to a protein encoded by one of the ORFs of the presentinvention can be used as a diagnostic agent. Agents which bind to aprotein encoded by one of the ORFs of the present invention can beformulated using known techniques to generate a pharmaceuticalcomposition.

5. EXAMPLES Example 1 Isolation of SEQ ID NO: 1 from a Human cDNALibrary

The novel nucleic acid of SEQ ID NO: 1 was obtained from a human cDNAlibrary prepared from fetal skin (Invitrogen), using standard PCR,sequencing by hybridization sequence signature analysis, and Sangersequencing techniques. The inserts of the library were amplified withPCR using primers specific for vector sequences flanking the inserts.These samples were spotted onto nylon membranes and interrogated witholigonucleotide probes to give sequence signatures. The clones wereclustered into groups of similar or identical sequences, and a singlerepresentative clone was selected from each group for gel sequencing.The 5′ sequence of the amplified insert was then deduced using thereverse M13 sequencing primer in a typical Sanger sequencing protocol.PCR products were purified and subjected to fluorescent dye terminatorcycle sequencing. Single-pass gel sequencing was done using a 377Applied Biosystems (ABI) sequencer. The insert of SEQ ID NO: 1 wasdescribed as a novel sequence in international publication WO03/(029405).

Example 2 Assemblage of SEQ ID NO: 2

The nucleic acid (SEQ ID NO: 2) of the invention was assembled fromsequences that were obtained from a cDNA library by methods described inExample 1 above, and in some cases obtained from one or more publicdatabases. The final sequence was assembled using the EST sequences asseed. Then a recursive algorithm was used to extend the seed into anextended assemblage, by pulling additional sequences from differentdatabases (i.e. Nuvelo's database containing EST sequences, dbESTversion 124, gbpri 124, and UniGene version 124) that belong to thisassemblage. The algorithm terminated when there were no additionalsequences from the above databases that would extend the assemblage.Inclusion of component sequences into the assemblage was based on aBLASTN hit to the extending assemblage with BLAST score greater than 300and percent identity greater than 95%.

Using PHRAP (Univ. of Washington) or CAP4 (Paracel), a full-length genecDNA sequence and its corresponding protein sequence were generated fromthe assemblage. Any frame shifts and incorrect stop codons werecorrected by hand editing. During editing, the sequence was checkedusing FASTY and BLAST against Genbank (i.e. dbEST version 124, gbpri124, UniGene version 124, Genpept release 124). Other computer programswhich may have been used in the editing process were phredphrap andConsed (University of Washington) and ed-ready, ed-ext and cg-zip-2(Hyseq, Inc.). The full-length nucleotide and amino acid sequences areshown in the Sequence Listing as SEQ ID NOS: 2 and 4, respectively.

In order to express GIPF (SEQ ID NO: 4), the full-length GIPF DNA wasPCR amplified from Marathon-ready cDNA libraries (Clontech). The primaryPCR product was further amplified using nested PCR primers thatgenerated GIPF polypeptide when expressed in suitable cell lines, asdescribed below.

Example 3 Expression of GIPF in Murine and Human Tissues

A. Tissue Distribution of GIPF mRNA:

FIG. 2 shows the relative expression of GIPF mRNA that was derived fromhuman (A) and murine (B) tissues.

Total mRNA was derived from the tissues indicated in FIG. 2 according tothe protocol provided by the manufacturer (Qiagen, Valencia, Calif.).The RNA was subjected to quantitative real-time PCR (TaqMan) (Simpson etal., Molec Vision 6:178-183 (2000)) to determine the relative expressionof GIPF in the tissues shown. The forward and reverse primers that wereused in the PCR reactions of human RNA were: 5′ GACCATGCTGCCTGCTCTGACAC3′ (forward; SEQ ID NO: 29), and 5′ CACCCGCCTCCTTGCTCTCC 3′ (reverse;SEQ ID NO: 30), respectively; and the forward and reverse primers thatwere used in the PCR reactions of murine RNA were: 5′GGGGGAGACCACACCACCTGCT 3′ (SEQ ID NO: 31), and 5′TTGGACCTCGGCTCCTTGCTGTTC 3′ (SEQ ID NO: 32), respectively. DNA sequencesencoding Elongation Factor 1, β-actin, and ATP synthase 6 were used as apositive control and normalization factors in all samples. All assayswere performed in triplicate with the resulting values averaged.

The Y axis shows the number of copies of GIPF mRNA per cell assumingthat each cell has 400,000 mRNA transcripts of a median length of 1.2Kb, and that 2% of the total RNA in a cell is mRNA. FIG. 2 shows thatGIPF mRNA is expressed at low levels in all the tissues tested. Thehighest levels of GIPF mRNA were seen in mouse skin, lung, ovary andbrain, and in human small intestine, skin, skin, ovary, testis, andbreast.

B. Tissue Distribution of GIPF Protein:

Expression of GIPF in human tissue samples was detected using rabbitpolyclonal anti-GIPF antibodies (Table 1). The rabbit polyclonalantibodies were generated by immunizing rabbits with a peptide that waspredicted to be immunogenic, and having amino acid sequence Glu Ser LysGlu Ala Gly Ala Gly Ser Arg Arg Arg Lys Gly Gln (SEQ ID NO: 67).Anti-GIPF antibody was affinity purified from rabbit serum using GIPFpeptide coupled to Affi-Gel 10 (Bio-Rad), and stored inphosphate-buffered saline with 0.1% sodium azide. Tissue samples ofadrenal gland, bladder, breast, colon, kidney, liver, lung, ovary,pancreas, placenta, prostate, skin, small intestine, spleen, stomach,testis, thyroid, tonsil and uterus were prepared for immunohistochemicalanalysis (IHC) (LifeSpan Biosciences, Inc., Seattle, Wash.) by fixingtissues in 10% formalin, embedding in paraffin, and sectioning usingstandard techniques. Sections were probed using the GIPF-specificantibody and visualized with a biotin-conjugated anti-rabbit secondaryusing AEC as substrate.

The cellular localization of GIPF in human tissues is shown in Table 1.The most intense staining was seen in the cytoplasm of a subset ofpancreatic islet cells and in the cytoplasm of intraepithelialneuroendocrine cells of the intestine and stomach. Prominent stainingwas also present in the adrenal cortex, gastric foveolar epithelium, andrenal tubular epithelium. Lymphocytes were frequently positive andshowed predominantly nuclear staining. In the skin, focal positivity waspresent in the stratum granulosum and in pilosebaceous units. A few celltypes showed less intense cytoplasmic and nuclear staining, includingrespiratory epithelium, type II pneumocytes, prostatic epithelium, andbreast epithelium. Focal faint nuclear staining was present inhepatocytes, colonic epithelium, placental trophoblasts, breastepithelium, ovary, and thyroid follicular epithelium.

Ganglion cells showed blush staining, while other cell types includingglandular epithelium, smooth muscle, endothelium, intravascularneutrophils, and fibroblasts tested negative for GIPF.

TABLE 1 Organ/Tissue Subcellular Location Staining Adrenal gland,epithelium Nuclear and cytosolic Focal, moderate Bladder, epitheliumNuclear and cytosolic Light Breast, epithelium Nuclear and cytosolicModerate Colon, epithelium Nuclear and cytosolic Moderate Colon,neuroendocrine Nuclear and cytosolic Faint cells Kidney cortex,glomeruli Nuclear Light Kidney cortext, epithelium Cytosolic ModerateKidney medulla, glomeruli Nuclear Strong Kidney medulla, epitheliumCytosolic Moderate Liver, hepatocytes Nuclear Moderate Lung, respiratoryNuclear and cytosolic Moderate epithelium Lung, type II pneumocytesPredominantly nuclear Moderate Lung, alveolar Predominantly nuclearModerate macrophages Ovary, epithelium Nuclear Moderate Pancreas, isletsof Cytosolic Strong Langerhans Placenta, trophoblasts Nuclear LightProstate, epithelium Nuclear Moderate Skin, epidermal layer CytosolicFocal Small intestine, Cytosolic, punctate Strong neuroendocrine cellsnuclear Small intestine, Predominantly nuclear Moderate inflammatorycells Spleen, lymphocytes Predominantly nuclear Strong Stomach,neuroendocrine Nuclear and cytosolic Focal, moderate to cells strongStomach, epithelium Nuclear and cytosolic Moderate to strong Testis,Leydig cells Cytosolic Light, sporadic Thymus, lymphocytes Nuclear andcytosolic Moderate Thyroid, follicular Nuclear Light epithelium Tonsil,lymphocytes Nuclear and cytosolic Moderate Uterus, endometrial stromaNuclear Moderate, sporadic

Example 4 Transgenic GIPF Animals A. Construction of the GIPF-KI Vector.

The construction of the transgene GIPF-knock-in (GIPF-KI) vector (FIG.5A) was performed according to the method described below, and depictedin FIGS. 5B-5R.

The DNA encoding the mouse Immunoglobulin kappa Constant region (IgCκ)and the proximal region was obtained by amplification of two fragmentsas follows:

FIG. 5B Preparation of IQCκ Fragment 1.

The forward (igkc1; SEQ ID NO: 34) and reverse (igkc2; SEQ ID NO: 35)primers for PCR were synthesized based on the sequence of the mouseImmunoglobulin kappa Constant region (IgCκ) and the proximal region thatwas obtained from GenBank (gi: V00777; SEQ ID NO: 33), and used toamplify the DNA that encodes fragment 1 of the IgCκ fragment 1. igkc1:ATCTCGAGGAACCACTTTCCTGAGGACACAGTGATAGG (SEQ ID NO: 34) was prepared byadding a Xho I recognition sequence at 5′ end site, and igkc2:ATGAATTCCTAACACTCATTCCTGTTGAAGCTCTTGAC (SEQ ID NO: 35) was prepared byadding an EcoR I recognition sequence at 5′ end site. PCR was carriedout using 25 ng of a clone of pBluescript SK II (+), which contains themouse Constant and Joining regions (WO 00/10383), and served astemplate. The PCR product was digested with restriction enzymes EcoR Iand Xho I and ligated into pBluescript II KS(−) vector (Stratagene) thatwas pre-digested with the restriction enzymes EcoR I and Xho I. Theresulting plasmid pIgCκA contained the designated cDNA sequence of themouse IgCκ fragment 1 with no substitution in nucleotide sequence withinthe region between Xho I and EcoR I.

FIG. 5C Preparation of IgCκ Fragment 2.

The forward (igkc3; SEQ ID NO: 36) and reverse (igkc4; SEQ ID NO: 37)primers for PCR were synthesized based on the sequence of the mousedownstream region of Immunoglobulin kappa Constant region (IgCκ) thatwas obtained from GenBank (gi: V00777; SEQ ID NO: 33), and used toamplify the DNA that encodes IgCκ fragment 2. Igkc3:ATGAATTCAGACAAAGGTCCTGAGACGCCACC (SEQ ID NO:36) was prepared by addingan EcoR I recognition sequence at 5′end site, and igkc4:ATGGATCCTCGAGTCGACTGGATTTCAGGGCAACTAAACATT (SEQ ID NO:37) was preparedby adding BamH I, Xho I, and Sal I recognition sequence at 5′ end site.PCR was carried out using 25 ng of the clone of pBluescript SK II (+)that contains the mouse Constant and Joining regions, and served astemplate (WO 00/10383). The PCR product was digested with restrictionenzymes EcoR I and BamH I and ligated into the pIgCκA vector (seeabove), pre-digested with the restriction enzymes EcoR I and BamH I. Theresulting plasmid pIgCκAB contained the designated cDNA fragments 1 and2 derived from the mouse Immunoglobulin Constant region with nosubstitution in nucleotide sequence within the region between EcoR I andBamH I.

FIG. 5D Insertion of Puromycin Gene into pIgCκAB

The Lox-P Puro plasmid (WO 00/10383) was digested with restrictionenzymes EcoR/and Xho I and treated with T4DNA polymerase. The resultingfragment was ligated into pIgCκAB vector (see above) pre-digested withthe restriction enzyme Sal I and treated with T4DNA polymerase. Afterverifying the connecting regions between pIgCκAB and the Lox-P Purofragment, the plasmid pIgCκABP was obtained.

FIG. 5E Insertion of IRES cDNA into pIgCκ ABP

The following forward (iresfw; SEQ ID NO:38) and reverse (iresrv; SEQ IDNO: 39) primers for PCR were synthesized based on the sequence of theIRES region of the pIREShyg plasmid (Clontech). iresfw:ATGAATTCGCCCCTCTCCCTCCCCCCCCCCTA (SEQ ID NO: 38) was prepared by addingan EcoR I recognition sequence at 5′ end site, and iresrv:ATGAATTCGTCGACTTGTGGCAAGCTTATCATCGTGTT (SEQ ID NO: 39) was prepared byadding EcoR/and Sal I recognition sequences at the 5′end site. PCR wascarried out using 150 ng of pIREShyg plasmid (Clontech) as a template.The PCR product was digested with the restriction enzyme EcoR I andligated into the pGEM-T vector (Promega) which had been digested inadvance with the restriction enzyme EcoR I. The plasmid IRES-Sal/pGEMwas obtained that contained the designated cDNA sequence with nosubstitutions in nucleotide sequence. The IRES-Sal/pGEM plasmid wasdigested with restriction enzyme EcoR/and ligated into the pIgCκABPplasmid (see above) which had been digested in advance with therestriction enzyme EcoR I. After verifying the sequence of connectingregions between pIgCκ ABP and IRES-Sal, the plasmid pIgCκ ABP IRES wasobtained.

FIG. 5F Construction of PACκ Sal Plasmid

The IgCk KO vector (WO 00/10383) was digested with restriction enzymeSac II and then partially digested with restriction enzyme EcoRI. A 14.6Kb band that lacked the LoxP-PGK Puro region was isolated and ligatedwith a SaclI/EcoRI compatible linker generated by annealing thefollowing two oligonucleotides (Sal1 plus and Sal1 minus), in order toreplace the LoxP-PGKPuro region with a Sal I restriction site. Aftersequence verification the pΔCκSal plasmid was obtained. Sal1 plus: 5′AGTCGACA 3′ (SEQ ID NO: 40) and Sal1 minus: 5′AATTTGTCGACTGC 3′ (SEQ IDNO: 41).

FIG. 5G Construction of PKIκ Plasmid

The pIgCκABP IRES plasmid was digested with the restriction enzyme Xho Iand the resulting fragment consisting of C region, IRES andloxP-Puromycin was ligated with pΔCκSal vector (see above) which hadbeen digested in advance with the restriction enzyme Sal I. Aftersequence verification the PKIκ plasmid was obtained.

FIG. 5H Preparation of pIgCκΔ IRES Fragment

The pIgCκABPIRES plasmid was partially digested with restriction enzymesEcoR I and Bgl II and the resulting pIgCκΔ IRES fragment that lacked aportion of the IRES gene was isolated.

FIG. 5I Preparation of Mouse P2 Promoter Fragment by PCR

The following primers for PCR were synthesized based on the sequence ofthe mouse Immunoglobulin kappa promoter obtained from GenBank (gi:aj231225; SEQ ID NO: 42). P2F:CCCAAGCTTTGGTGATTATTCAGAGTAGTTTTAGATGAGTGCAT (SEQ ID NO: 43) wasprepared by adding a Hind III recognition sequence at 5′ end site, and

P2R:ACGCGTCGACTTTGTCTTTGAACTTTGGTCCCTAGCTAATTACTA (SEQ ID NO: 44) wasprepared by adding a Sal I recognition sequence at 5′ end site. PCR wascarried out using 25 ng of mouse genomic DNA as a template (genomic DNAfrom TT2F ES cells). The PCR product was digested with restrictionenzymes Hind III and Sal I and ligated into pBluescript II KS-vector(Stratagene) which had been digested in advance with the restrictionenzymes Hind III and Sal I. After sequence verification, the resultingplasmid was digested with the restriction enzymes Hind III and Sal and aHind III-Sal I fragment containing the mouse P2 promoter fragment wasisolated.

FIG. 5J. Preparation of Partial Cκ polyA Fragment by PCR

The following primers for PCR were synthesized based on sequence of themouse Immunoglobulin kappa polyA region obtained from GenBank(gi:v00777) PPF:ACGCGTCGACGCGGCCGGCCGCGCTAGCAGACAAAGGTCCTGAGACGCCACCACCAGCTCCCC (SEQ ID NO: 45) was prepared by adding Sal I, Fse I, andNhe I recognition sequence at 5′ end site, and PPR:GAAGATCTCAAGTGCAAAGACTCACTTTATTGAATATTTTCTG (SEQ ID NO: 46) was preparedby adding a Bgl II recognition sequence at 5′ end site. PCR was carriedout using 25 ng of mouse genomic DNA as a template (genomic DNA fromTT2F ES cells). The PCR product was digested with restriction enzymesSal I and Bgl II and ligated into the psp72 vector (Promega KK) whichhad been digested in advance with the restriction enzymes Sal I and BglII. After sequence verification, the purified plasmid was digested withthe restriction enzymes Sal I and Bgl II, to generate the “partial CkpolyA fragment”.

FIG. 5K. Preparation of Total Cκ PolyA Fragment by PCR.

The following primers for PCR were synthesized based on sequence of themouse Immunoglobulin kappa polyA region obtained from GenBank(gi:v00777): TPF: GGAATTCAGACAAAGGTCCTGAGACGCCACCACCAGCTCCCC (SEQ ID NO:47) was prepared by adding an EcoR I recognition sequence at 5′ endsite, and TPR: CCCAAGCTTGCCTCCTCAAACCTACCATGGCCCAGAGAAATAAG (SEQ ID NO:48) was prepared by adding a Hind III recognition sequence at 5′ endsite. PCR was carried out using 25 ng of mouse genomic DNA as a template(genomic DNA from TT2F ES cells). The PCR product was digested withrestriction enzymes EcoR I and Hind III and ligated into pBluescript IIKS-vector (Stratagene) which had been digested in advance with therestriction enzymes EcoR I and Hind III. After sequence verification theplasmid was digested with the restriction enzymes EcoR I and Hind III,to generate the “total Ck polyA fragment”.

FIG. 5L. Construction of DNA Fragment A that Consists of Total Cκ polyAFragment, P2 Promoter Fragment, and Partial Ck polyA Fragment.

The “total Cκ poly A fragment”, “P2 promoter fragment”, and “partial Cκpoly A fragment” generated as described above were ligated in thedescribed order into pBluescript II KS-vector (Stratagene) which hadbeen digested in advance with the restriction enzymes EcoR I and Bgl II.After sequence verification, the purified plasmid was digested with therestriction enzymes EcoR I and Bgl II, to generate “DNA fragment A”.

FIG. 5M. Construction of PlgCκΔ IRES ProA Plasmid

“DNA fragment A” was ligated into the “pIgCκ A IRES fragment” isolatedas described above. After sequence verification the plasmid pIgCκ A IRESProA was obtained.

FIG. 5N. Construction of Cκ P2H Plasmid

pIgCκΔ IRES ProA plasmid was digested with Xho I and the main fragmentthat contained the upstream genomic region of mouse IgCκ, mouse IgCκ,DNA fragment A and Lox-P Puro thus isolated, was ligated with pΔCκ Salwhich had been digested in advance with the restriction enzyme Sal I.After sequence verification the plasmid Cκ P2H was obtained.

FIG. 5O. Construction of Cκ 5′ Genomic Plasmid

The following primers for PCR were synthesized based on sequence of aDNA segment containing the mouse immunoglobulin kappa J and C regiongenes obtained from GenBank (gi: v00777). 5GF:ATAAGAATGCGGCCGCCTCAGAGCAAATGGGTTCTACAGGCCTAACAACCT (SEQ ID NO: 49) wasprepared by adding a Not I recognition sequence at 5′end site, and 5GR:CCGGAATTCCTAACACTCATTCCTGTTGAAGCTCTTGACAATGG, (SEQ ID NO: 50) wasprepared by adding an EcoR I recognition sequence at 5′end site. PCR wascarried out using 25 ng of mouse genomic DNA (genomic DNA from TT2F EScells as a template). The PCR product was digested with restrictionenzymes Not I and EcoR I and ligated with pBluescript II KS-vector(Stratagene) which had been digested in advance with the restrictionenzymes Not I and EcoR I. After sequence verification, the Cκ 5′ genomicplasmid was obtained.

FIG. 5P. Construction of Cκ P2 KIΔ DT Plasmid

The Cκ P2H plasmid was digested with EcoR I and Xho I and a 11 Kb wasobtained and was ligated into the Cκ 5′ genomic plasmid which had beendigested in advance with the restriction enzymes EcoR I and Xho I. Aftersequence verification the Cκ P2 KIΔ DT plasmid was obtained.

FIG. 5Q. Construction of Cκ P2 KI Vector

The DT-A fragment was isolated from the pKIκ plasmid using restrictionenzymes Xho I and Kpn I, and was ligated into the Cκ P2 KIΔ DT plasmidwhich has been digested in advance with the restriction enzymes Xho Iand Kpn I. After sequence verification the CKP2 KI vector was obtained.

FIG. 5R. Assembly of the GIPF-KI Vector.

A GIPF cDNA fragment was amplified using the following primers for PCR,which were synthesized based on the sequence of human GIPF cDNA (SEQ IDNO: 2). SA3F: ACGCGTCGACCCACATGCGGCTTGGGCTGTGTGT (SEQ ID NO: 51) wasprepared by adding a Sal I recognition sequence and Kozak sequence at 5′end site, and SA3R: ACGCGTCGACGTCGACCTAGGCAGGCCCTG (SEQ ID NO: 52) wasprepared by adding a Sal I recognition sequence at 5′ end site.PCR was carried out using a pool of Marathon-Ready cDNA (fetal skin andfetal lung, BD Biosciences CLONTECH) as a template. The PCR product wasdigested with restriction enzyme Sal and ligated with pBluescript IIKS-vector (Stratagene) which has been digested in advance with therestriction enzyme Sal I. After sequence verification a clone wasobtained and verified to contain the correct GIPF cDNA sequence with nosubstitution in nucleotide sequence. The clone was digested withrestriction enzyme Sal I, and the GIPF cDNA fragment was purified andligated into CK P2 KI vector, which had been digested in advance withthe restriction enzyme Sal I. After sequence verification, the GIPF-KIvector, was obtained (FIG. 5A).

B. Generation of GIPF-KI Transgenic Mice

General procedures for obtaining mouse embryos, cultivation, injectionof the ES cells into the embryos, transplantation to the uteri of fostermothers were carried out in accordance with the method described inAizawa Shinichi, “Biomanual Series 8, Gene Targeting”, published byYodosha, 1995.

The GIPF-KI vector was linearized with Not I and transferred intoC57BL/6×CBA F1 derived mouse TT2F ES cells ((Uchida, 1995), Lifetechoriental) by electroporation according to the method described byShinichi Aizawa, “Biomanual Series 8, Gene Targeting”, published byYodosha, 1995. The electroporated ES cells were suspended in 20 ml of ESmedium [DMEM (GIBCO), 18% FCS (GIBCO), 0.1 mM 2-mercaptoethanol (GIBCO),1000 U/ml LIF (leukemia inhibitory factor, CHEMICON International,Inc.)] and inoculated into two 100 mm tissue culture plastic plates(Corning) into which feeder cells (Invitrogen) were seeded in advance.After one day, the medium was replaced with a medium containing 0.75g/ml of puromycin (Sigma). Seven to nine days thereafter, a total of 119colonies formed were picked up. Each colony was grown up to confluencein a 12-well plate, and then four fifths of the culture was suspended in0.2 ml of cryopreservation medium [ES medium+10% DMSO (Sigma)] andstored frozen at −80° C. The remaining one fifth was inoculated into a12-well gelatin coated plate and cultured for 2 days. Then, genomic DNAwas isolated using the Puregene DNA Isolation Kit (Gentra System).Genomic DNA isolated from puromycin resistant TT2F cells was digestedwith restriction enzyme EcoR I (Takara Shuzo) and then subjected to 0.8%agarose gel electrophoresis. Separated DNA fragments were transferred toa membrane (GeneScreen, NEN™ Life Science Products) and thenhybridization was carried out using the DNA fragment as probe preparedfrom 3′ region of IgJκ-Cκgenomic DNA (Xho I-EcoR I, 1.3 Kb (SEQ ID NO:67): WO 00/10383, Example No. 48). The band pattern of untargeted ESclone shows one band of MW of about 15 Kb and targeted ES clone showstwo bands of MW of about 15 Kb and 13.4 Kb (FIG. 6). Two out of 48targeted ES clones #10, 12 were selected after Southern analysis (rateof homologues recombination was about 4.2%). The selected ES clones werealso tested by karyotype analysis according to the method described byShinichi Aizawa, “Biomanual Series 8, Gene Targeting”, published byYodosha, 1995. Two ES clones #10, 12 that showed normal karyotype wereused for implantation into embryos.

The cells in a frozen stock of the targeted ES cell clones #10, 12 werethawed, started to culture and injected into 8-cell stage embryosobtained by mating a male and a female mouse of Immunoglobulin heavychain knock out mouse strain (Tomizuka et. al. Proc. Natl. Acad. Sci.USA, 97: 722-727, 2000); the injection rate was 10-12 cells per embryo.After the embryos were cultured overnight in the medium for ES cells todevelop into blastocysts, about ten of the TT2F cell-injected embryoswere transplanted to each side of the uterus of a foster mother ICRmouse (CREA JAPAN, INC.), which had been subjected to a pseudopregnanttreatment for 2.5 days. As a result of transplantation of a total of 120injected embryos, 24 offspring mice were born. Chimerism in theoffspring was determined by the extent of TT2F cell-derived agouti coatcolor (dark brown) in the host embryo (ICR)-derived albino coat color.Out of the 24 offspring, 11 mice (knock-in mice) were recognized to havepartial agouti coat color, indicating the contribution of the ES cells.Genomic DNA isolated from the tails of Knock-in mice was used for PCRanalysis. The following two primers for PCR were synthesized based onsequence of GIPF-KI vector: SACF: CTGACTAGACTCTATCTTGC (SEQ ID NO: 53),and SACR: CCACGGAGACCACTCGCTCATT (SEQ ID NO: 54).

PCR was carried out using 25 ng of mouse tail genomic DNA as a template.The resulting reaction solution was subjected to 0.8% agarose gelelectrophoresis, and 606 bp band was detected. Normal TT2F cell cloneswere used for control chimeric mice production.

Mice were kept under a 12/12-hour dark/light cycle (lights on at 8:00am) and received 5 μm filtered water and CE-2 food (CLEA JAPAN, INC.) adlibitum. Male mice were housed individually after weaning period.

Example 5 Evaluation of the Biological Activity of GIPF Using TransgenicGIPF-KI Mice

The gross pathological changes and the histological changes of the smallintestine and colon from the transgenic mice described above wasevaluated as follows.

GIPF transgenic KI mice demonstrated auxetic growth of small intestinestarting at the age of 4 weeks and significant abdominal distensionduring development. FIG. 7 shows that 15 week old GIPF transgenic KImice had marked intestinal distension and increased small intestinalmass when compared to the corresponding control KI mouse.Histo-pathological evaluation, using hematoxylin and eosin (H&E)staining (Issacson, P. G., and Wright, D. H., 1983) was done on paraffinembedded sections (5 μm thick) of various tissues including liver,spleen, lung, kidney, heart, small intestine and large intestine. H&Esections of small intestine were shown in FIG. 8 (low magnification) andFIG. 9 (high magnification). A histopathology report was provided byIDEXX Laboratories, Inc. and is described below.

The only significant difference between the histologic appearance of thetissues from the control and the knock-in mouse was found in the smallintestine. This change consisted of a marked, diffuse thickening of themucosa by crypt epithelial hyperplasia with a marked increased in cryptlength and complexity of branching. The crypts were lined by plumpcolumnar epithelial cells with basophilic cytoplasm and basally-locatedlarge ovoid heterochromatic nuclei with frequent mitoses. Numerousapoptotic bodies were scattered throughout the crypt epithelium,suggesting an increased rate of cell turnover. The crypt epithelialcells also commonly differentiated into both Paneth cells andmucus-secreting goblet cells throughout the length of the crypts. Thevillous epithelium was not significantly altered. Similar changes wereseen in the small intestinal mucosa associated with the Peyer's patch.The intestinal mucosa normally externally lines and may form smallinvaginations into the lymphoid tissue of the Peyer's patch. In theGIPF-KI mice, the hyperplastic changes were also seen on the surface andin invaginations, where they were associated with mild acuteinflammation and accumulations of necrotic cells within crypts, i.e.crypt microabscesses. This Peyer's patch was sectioned tangentiallytherefore the amount and character of lymphoid tissue was difficult toevaluate. However, there were small numbers of plasma cells indicatingtransformation of B-lymphocytes into antibody-producing cells. Therewere no other visible alterations in lymphoid or inflammatory cellpopulations in the intestine or other tissues.

To measure intestinal epithelial cell proliferation in KI mice,immunohistochemistry using monoclonal rat anti-mouse Ki67 antigen (DakoLtd., High Wycombe, UK) was performed on paraffin embedded sections ofsmall intestine of control and GIPF KI mice according to manufacturer'sinstruction and the method previously described (Scholzen, T. et al.2000). As shown in FIG. 10, GIPF KI mice demonstrated increased Ki67positive epithelial cells in the small intestine suggesting increasedproliferation by GIPF protein expression.

Three of the GIPF-KI mice were harvested at 12 months. These 12month-old mice displayed the typical abdominal distention and increasedintestinal mass seen in younger animals. H&E sections were prepared invarious tissues including spleen, liver, adrenal, kidney, thymus, heart,lung, small intestine, large intestine, stomach and brain.

Histology of sections from the small intestine of the 12 month oldGIPF-KI mice showed that GIPF had induced a marked increase in cryptlength and to the same extent as that seen in 15 weeks old GIPF-KIanimals. In addition, histological analysis of sections from otherorgans revealed that over the extended period of 12 months GIPF did notdisplay any apparent tumorigenic activity. Spontaneous tumorigenesis wassometimes observed in some mice irregardless of whether the mice werenormal or KI transgenic animals. A low incidence of hepatic adenomas wasobserved in 12 month-old control mice.

Example 6 The GIPF Adenoviral Vector

The GIPF cDNA (SEQ ID NO: 2) was cloned into pAdenoVator CMV5-Intronusing NheI and XbaI sites in multi-cloning sites (MCS) to generateV5His6 tagged GIPF recombinant adenovirus. pAdenoVator-CMV5-Intron wasobtained by modification of pAdenoVator CMV5-IRES-GFP (Qbiogene,Carlsbad, Calif., U.S.A). pAdenoVator-CMV5-IRES-GFP was digested withSpeI to remove its MCS, IRES and GFP and ligated with PCR amplifiedIntron-MCS-V5His-BGH polyA from pcDNA/Intron vector using primers:5′-CACCCCTAGGTCAATATTGGCCATTAGC-3′ (SEQ ID NO: 55) and5′-CACCCCT-AGGTAGGCATCCCCAGCATGC-3′ (SEQ ID NO: 56).

Transformation of linearized transfer vector into bacterial cells,BJ5183, (Qbiogene, Carlsbad, Calif., U.S.A) which carry AdEasy-1 plasmidthat encode Adenovirus-5 genome (E1/E3 deleted) was performed byelectroporation according to the manufacturer's instructions.Recombinant adenovirus was generated and amplified in QBI-293A cells(Qbiogene, Carlsbad, Calif., U.S.A) and purified by CsCl banding aspreviously described (Garnier, A., J. Cote et al. 1994). Recombinantprotein expression by 293A cells that had been infected with therecombinant adenovirus was measured by Western analysis using anti-V5antibody (Invitrogene Inc., Carlsbad, Calif.). The titer of CsClpurified recombinant viruses was measured using the Adeno-X rapid titerkit (BD biosciences, Palo Alto, U.S.A.) according to the manufacturer'sprotocols. Briefly, a viral stock was tested by infecting 293A cellswith serial dilutions of the recombinant adenovirus stock followed byfixation and staining of the transduced cells with mouse anti-hexonantibody 48 hours after infection. The signal was detected with a goatanti-mouse antibody conjugated to horseradish peroxidase and developedwith metal-enhanced 3,3′-diaminobenzidine tetrahydro-chloride (DAB).

Example 7 Administration of GIPF Adenovirus as a Model to Evaluate theBiological Activity of GIPF

The GIPF recombinant adenovirus was administered to normal mice todetermine the effect of GIPF on the intestinal and colonic epithelium,and to confirm that the phenotype observed in the GIPF transgenic micecould be established in a non-transgenic animal. Prior to injection ofadenovirus, BALB/C mice, 6-8 weeks of age, were anesthetized usingisoflurane. 1×10¹⁰ viral particles per mouse were injected throughretro-orbital vein. The same titer of control virus (empty virus) or PBSalone were used as controls. Mice were sacrificed on day 3 or day 5after virus injection (n=3 for all group). 4 hours before sacrifice, 1mg of bromodeoxyuridine (BrdU) was injected intraperitoneally (IP) todetermine in vivo proliferation of epithelial cells. Various tissuesincluding small intestine, colon, spleen, liver and bone marrow werecollected and fixed in formaline. Paraffin embedded sections werestained with hematoxylin and eosin (H&E) for histological evaluation.Sections were also processed for BrdU immunohistochemistry according tothe manufacturer's instruction (Oncogene Research product, Boston,U.S.A.) as previously described (McKinley, J. N. et al. 2000).Immunohistochemistry using monoclonal rat anti-mouse Ki67 antigen (DakoLtd., High Wycombe, UK) was also performed to assess the proliferationof intestinal epithelial cells according to manufacturer's instructionand the method previously described (Scholzen, T. et al. 2000).

H&E staining of sections from the small intestine that had beensacrificed 3 days following the adenovirus injection (FIG. 11) show thatthe small intestine of mice that had received the GIPF adenovirus wassignificantly altered, and displayed the same histologicalcharacteristics seen in the GIPF transgenic mice (FIGS. 8 and 9). Thehistological changes caused by GIPF included a marked, diffusethickening of the mucosa by crypt epithelial hyperplasia with a markedincreased in crypt length and complexity of branching. The crypts werelined by plump columnar epithelial cells with basophilic cytoplasm andbasally-located large ovoid heterochromatic nuclei with frequentmitoses. The crypt epithelial cells also commonly differentiated intoboth Paneth cells and mucus-secreting goblet cells throughout the lengthof the crypts. The effect of GIPF on crypt epithelial proliferation wasfurther enhanced in 5 days after virus injection which was shown in FIG.12. To evaluate the effect of GIPF on the proliferation of intestinalepithelial cells, BrdU incorporation and Ki67 immuno-staining wereperformed on small intestinal sections of control and mice that hadreceived GIPF adenovirus. As shown in FIGS. 13 and 14, the mice that hadreceived the GIPF adenovirus had small intestinal crypts that hadsignificantly more BrdU and Ki67 positive cells, respectively. Thebiological effect of GIPF on the proliferation of crypt epithelial cellswas also observed at a lower viral dose of 1×10⁹ viral particles permouse (FIG. 15). In addition to the effect seen in the small intestine,GIPF also induced crypt epithelial hyperplasia with a marked increasedin crypt length and an increased number and size of Goblet cells in thecolon (FIG. 16).

Example 8 Expression Vectors Encoding GIPF and GIPF Analogs

The cDNA encoding GIPF (SEQ ID NO: 3) was cloned into pcDNA/Intronvector using KpnI and XbaI sites to generate wild type andcarboxy-terminal V5His6-tagged GIPF (SEQ ID NO: 5). The mammalianexpression vector pcDNA/Intron was obtained by genetically modifying thepcDNA3.1TOPO vector (Invitrogene Inc., Carlsbad, Calif.) by introducingan engineered chimeric intron derived from the pCI mammalian expressionvector (Promega, Madison, Wis.). pCI was digested with BGIII and KpnI,and the intron sequence was cloned into pcDNA3.1, which had beendigested with BgIII and KpnI. The GIPF ORF of SEQ ID NO: 2 (SEQ ID NO:3) was first cloned into pcDNA3.1N5His-TOPO (Invitrogen) by PCR usingthe following forward 5′ CACCATGCGGCTTGGGCTGTCTC 3′ (SEQ ID NO: 57)reverse 5′ GGCAGGCCCTGCAGATGTGAGTG 3′ (SEQ ID NO: 58), and the KpnI-XbaIinsert from pcDNA 3.1N5His-TOPO that contains the entire GIPF ORF wasligated into the modified pcDNA/Intron vector to generate pcDNA/Intronconstruct.

Analogs of the full-length GIPF protein were generated as follows.Mutation of the predicted consensus furin cleavage sites (amino acid 28R/Q) of GIPF in pcDNA/Intron was performed by site directed mutagenesisusing primers 5′-GATCAAGGGGAAACAGCAGAGGCGGATCAG-3′ (SEQ ID NO: 59) and5′-CTGATCCGCCTCTGCTGTTTCCCCTTGATC-3′ (SEQ ID NO: 60). The GIPF deletionmutant (deleted amino acid residues 21-31) (SEQ ID NO: 16) was generatedusing the stitching method. The deletion was introduced by PCRamplification of two fragments using primers set1:5′-CACCGCTAGCCTCGAGAATTCACGCGTG-3′ (SEQ ID NO: 61) and phospho5′-GCTGATGGTGAGGTGCGTC-3′ (SEQ ID NO: 62), set2: phospho5′-ATCAGTGCCGAGGGGAGCCAG-3′ (SEQ ID NO: 63) and5′-GCCCTCTAGAGCGGCAGGCCCTGCAGATG-3′ (SEQ ID NO: 64) followed by ligationof the two fragments. The GIPF cDNA carrying deletion of amino acids21-31 was amplified by PCR using the forward and reverse primers of SEQID NO: 61 and 64, respectively, digested with NheI and XbaI, andsubcloned into pcDNA/Intron vector using NheI and XbaI sites in itsmulticloning sites. Sequences were confirmed for both mutants.

The thrombospondin (TSP) domain (nt 451 to nt 618 of the ORF of GIPF(SEQ ID NO: 13) was also cloned into pcDNA/Intron vector for mammalianexpression. The cDNA encoding the TSP domain was amplified by PCR usingNheI forward primer: CCGGCTAGCCACCATGGCGCAATGTGAAATGA (SEQ ID NO: 65)and NotI reverse primer: CCATGCGGCCGCCCTCCTCACTGTGCACCT (SEQ ID NO: 66).NheI and NotI restriction enzymes digested PCR product was ligated intoNheI and NotI digested pcDNA/Intron vector. To generate recombinantadenovirus, the TSP domain from PCR amplification described above wascloned into pAdenoVator-CMV5-Intron using NheI and NotI restrictionenzymes. The sequence of the PCR-amplified TSP domain was confirmed.

Other analogs that lack various portions of the furin-like cysteine-richregion of GIPF are described in Example 19.

The biological activity of the GIPF analogs described above was assessedin vivo and in vitro using methods described in the examples below. Thebiological activity of the GIPF analogs is assessed using the GIPFtransgenic model.

Example 9 Purification of Recombinant GIPF A. Expression andPurification GIPFt in Eukaryotic Cells:

V5-His-tagged GIPF (GIPFt) (SEQ ID NO: 5) was expressed in HEK293 andCHO cells and purified as follows:

A stable cell culture of HEK293 cells that had been transfected with theGIPF pcDNA/Intron construct comprising the DNA encoding theV5-His-tagged GIPF polypeptide (SEQ ID NO: 5) was grown in serum free293 free-style media (GIBCO). A suspension culture was seeded at celldensity of 1 million cells/ml, and harvested after 4-6 days. The levelof the V5-His-tagged GIPF that had been secreted into the culture mediumwas assayed by ELISA.

A stable cell culture of CHO cells that had been transformed with a pDEF2S vector comprising nucleotide sequence that encodes a V5-His taggedGIPF (SEQ ID NO: 5) was grown in serum free EX-CELL302 media (JRH). Theexpression vector contains DNA sequence that encodes DHFR, which allowsfor positive selection and amplification in the presence of methotrexate(MTX). The level of the V5-His-tagged GIPF that had been secreted intothe culture medium was assayed by ELISA.

The media containing the secreted GIPF protein was harvested and frozenat −80° C. The media was thawed at 4° C., and protease inhibitors, EDTAand Pefabloc (Roche, Basel, Switzerland) were added at a finalconcentration of 1 mM each to prevent degradation of GIPF. The mediawere filtered through a 0.22 μm PES filter (Corning), and concentrated10-fold using TFF system (Pall Filtron) with a 10 kDa molecular weightcut-off membrane. The buffers of the concentrated media were exchangedwith 20 mM sodium phosphate, 0.5M NaCl, pH 7. The addition of 0.5 M NaClin the phosphate buffer is crucial to keep full solubility of V5-Histagged GIPF at pH 7 during purification. Following ultrafiltration anddiafiltration, a mammalian protease inhibitor cocktail (Sigma) was addedto a final dilution of 1:500 (v/v).

A HiTrap Ni²⁺-chelating affinity column (Pharmacia) was equilibratedwith 20 mM sodium phosphate, pH 7, 0.5 M NaCl. The buffer-exchangedmedia was filtered with 0.22 μm PES filter and loaded ontoNi²⁺-chelating affinity column. The Ni²⁺ Column was washed with 10column volumes (CV) of 20 mM imidazole for 10 Column Volume and proteinwas eluted with a gradient of 20 mM to 300 mM imidazole over 35 CV. Thefractions were analyzed by SDS-PAGE and Western blot. Fractionscontaining V5-His tagged GIPF were analyzed and pooled to yield a GIPFprotein solution that was between 75-80% pure.

The buffer containing the GIPF protein isolated using the Ni²⁺ columnwas exchanged with 20 mM sodium phosphate, 0.3 M Arginine, pH 7 toremove the NaCl. NaCl was replaced with 0.3 M Arg in the phosphatebuffer to maintain full solubility of V5-His tagged GIPF protein duringthe subsequent purification steps. The GIPF protein isolated using theNi²⁺ column was loaded onto a SP Sepharose high performance cationexchange column (Pharmacia, Piscataway, N.J.) that had been equilibratedwith 20 mM sodium phosphate, 0.3 M Arginine, pH 7. The column was washedwith 0.1 M NaCl for 8 CV, and eluted with a gradient of 0.1 M to 1 MNaCl over 30 CV. Fractions containing V5-His tagged GIPF were pooled toyield a protein solution that was between 90-95% pure.

The buffer of the pooled fractions was exchanged with 20 mM sodiumphosphate, pH 7, 0.15 M NaCl, the protein was concentrated to 1 or 2mg/mL, and passed through a sterile 0.22 μm filter. The pure GIPFpreparation was stored at −80° C.

The protein yield obtained at the end of ach purification step wasanalyzed and quantified by ELISA, protein Bradford assay and HPLC. Thepercent recovery of GIPFt protein was determined at every step of thepurification process, and is shown in Table 2 below.

TABLE 2 Steps Step Recovery Overall Recovery Media 100% 100%Concentration/Diafiltration Ni-chelating Affinity 65% 65% SP CationExchange 80% 52% Final Formulation and 95% 49% filter

SDS-PAGE analysis of the purified GIPF protein was performed underreducing and non-reducing conditions, and showed that the V5-His taggedGIPF protein derived from both CHO and 293 cells exists as a monomer.GIPF protein is glycosylated and migrates on SDS-PAGE under non-reducingconditions with molecular weight (MW) of approximately 42 kDa. There isslight difference in the MW of the GIPF protein purified from CHO cellsand that purified from HEK293 cells. This difference may be explained bythe extent to which GIPF is glycosylated in different cell types.N-terminal sequence analysis showed that HEK293 cells produced two formsof the polypeptide: the dominant mature form (SEQ ID NO: 10) whichcorresponds to the GIPF protein of SEQ ID NO: 4 that lacks the signalsequence, and the mature form (SEQ ID NO: 12), which corresponds to theGIPF protein of SEQ ID NO: 4 that lacks both the signal peptide and thefurin cleavage sequence. The two forms separated well on the SP column,and were expressed at a ratio of mature to dominant mature forms ofapproximately 1:2.

The effect of NaCl and Arginine (Arg) on the solubility of the GIPFprotein at pH 7 was determined, and is shown in FIG. 17 A. It wasdetermined that in the absence of 0.3M Arg a 50% loss of protein wasincurred during the purification.

FIG. 17 B shows the solubility of purified protein in PBS (20 mM sodiumphosphate, 0.15 M NaCl, pH 7). GIPF protein remains in solution atconcentrations of up to 8 mg/mL at 4° C., pH7, for 7 days.

In summary, the purification of V5-His-tagged GIPF from cultures ofHEK293 or CHO cells was performed by 1) concentrating and diafilteringthe GIPF protein present in the culture media, 2) performingNi²⁺-chelating affinity chromatography, and 3) SP cation exchangechromatography. The purification process yields a GIPF protein thatis >90% pure. The overall recovery of the current purification processis approximately 50%. Addition of 0.5 M NaCl to the buffer during thepurification process of media diafiltration and Ni column is crucial tokeep GIPF fully soluble at pH 7. For binding GIPF onto the SP column,NaCl was removed, and 0.3 M Arg was added to maintain high solubilityand increase protein recovery. The addition of 0.5 M NaCl and 0.3 Argduring the first and second purification steps showed to increase theoverall recovery by at least from 25% to 50%.

The dominant mature and mature forms of the V5His-tagged GIPF were usedto test the biological activity of GIPF the in vivo setting described inExample 10. The protein purified by the method of this exampleconsistently induced significant proliferation of intestinal cryptepithelial cells, which underlies the distension of the small intestineof the mice that were administered the purified GIPF protein.

B. Expression and Purification of GIPFwt in Eukaryotic Cells:

The untagged, wild type GIPF protein (GIPFwt; SEQ ID NO: 4) wasexpressed and purified in a manner similar to that described for thetagged GIPF protein. A stable cell culture of HEK293 cells that had beentransfected with the pcDNA/Intron vector comprising the DNA (SEQ ID NO:3) encoding the full-length GIPF polypeptide (GIPFwt) (SEQ ID NO: 4) wasadapted to grow in suspension and grown in serum-free 293 free-stylemedium (GIBCO) in the presence of 25 μg/ml geneticin.

Cell culture growth in spinner: For small-scale production in spinners,an aliquot of a frozen stock of cells was grown and expanded in 293free-style media with addition of 0.5% Fetal Bovine Serum (FBS). Cellswere seeded and expanded in spinners at cell density of 0.3-0.5million/mL for each passage. When enough cells are accumulated and celldensity reaches 1 million cells/mL for production, the media wasexchanged with serum-free 293 free-style media to remove 0.5% FBS, andharvested after 6 days. The initial cell viability was between 80-90%and it decreased to 30% at the time of harvest. The level of GIFPwt thathad been secreted into the culture medium was assayed by ELISA andwestern. Growth of GIPFwt in the spinners yielded 1.2-1.5 mg/l.

Cell Culture Growth in Bioreactors—Fed-batch mode was used forlarge-scale production in bioreactors. A serum-free adapted suspensionculture of HEK293 cells was seeded at cell density of 0.2-0.4 million/mlwhen passage of cells. Cells were grown in serum free 293 free-stylemedium and expanded from 50-500 ml shake flasks to 20-50 stir tanks forinoculation of a 2001 and 5001 bioreactor. When enough cells wereaccumulated, the cells were inoculated into a bioreactor at a density of0.2-0.4 million cells/ml. When the cell density reached 1 millioncells/ml, vitamins and MEM amino acids (GIBCO) were added to boost andsupport the growth. Cells were harvested from the bioreactor after 6-7days when the cell viability had decreased to 25-30%. The level ofGIPFwt that had been secreted into the culture medium was assayed byELISA and western. Western analysis of the secreted GIPF showed that nodegradation of the protein had occurred. Western analysis was performedusing a purified anti-GIPF polyclonal antibody, and the detection of theprotein by ELISA was performed using a purified chicken anti-GIPFpolyclonal antibody as the capture antibody, and the rabbit anti-GIPFpolyclonal antibody as the detection antibody. The rabbit and chickenpolyclonal antibodies were raised against the whole protein. Growth ofGIPFwt in the bioreactors yielded 2.6-3 mg/l.

Ultrafiltration-Diafiltration—the medium containing the secreted GIPFwtprotein was harvested by centrifugation. Protease inhibitors 1 mM EDTAand 0.2 mM Pefabloc (Roche, Basel, Switzerland) were added to preventdegradation of GIPF. The medium was filtered through a 0.22 μm PESfilter (Corning), and concentrated 10-fold using TFF system (PallFiltron) or hollow-fiber system (Spectrum) with 10 kDa cut-off membrane.The buffer of the concentrated medium was exchanged with 20 mM sodiumphosphate, 0.3 M Arg, pH 7. The addition of 0.3 M Arg in the phosphatebuffer is crucial to keep GIPFwt fully soluble at pH 7 duringpurification. After ultrafiltration and diafiltration, a mammalianprotease inhibitor cocktail (Sigma) was added at 1:500 (v/v) dilution.

Q anion exchange chromatography—an anion exchange Q sepharose HP column(Amersham) was equilibrated with 20 mM sodium phosphate (NaP) buffer atpH7.0 and containing 0.3 M Arg. The 10-fold concentrated andbuffer-exchanged medium was filtered with 0.22 μm PES filter and loadedonto the Q sepharose column to bind impurities and nucleic acids.

SP cation exchange chromatography: the Q-sepharose flow throughcontaining GIPFwt was collected and loaded onto a cation exchange SPsepharose HP (Amersham), which bound the GIPF protein. The SP sepharosecolumn was washed with 15 column volumes (CV) of 20 mM NaP, 0.3 M Arg,0.1 M NaCl, pH 7, and GIPF was eluted with a gradient of 0.1 M to 0.7 MNaCl over 40 column volumes. The fractions were analyzed by SDS-PAGE andWestern blot. Fractions containing GIPFwt were analyzed and pooled. Thebuffer of the pooled fractions was exchanged with 20 mM sodiumphosphate, pH 7, 0.15 M NaCl. The purity of the purified protein wasdetermined to be 92-95% when analyzed by Coomassie staining of anSDS-gel. The protein was concentrated to 1 mg/ml, and passed through asterile 0.22 μm filter and stored at −80° C.

The yield obtained at the end of each step in the purification processwas quantified by ELISA and by the Bradford assay, and the percentrecovery of GIPF protein was calculated as shown in Table 3.

TABLE 3 Steps Step Recovery Overall Recovery Media 100% 100% Concentration/Diafiltration Q Anion Exchange 95% 95% SP Cation Exchange75% 71% Final Formulation and filter 98% 70% 48% (dominant mature formonly)

The endotoxin level of the final formulated GIPF protein solution wasanalyzed using chromogenic LAL (Limulus Amebocyte Lysate) assay kit(Charles River), and determined to be 0.24 EU per mg of GIPF.

C. Expression and Purification GIPFt in Yeast:

GIPFt was expressed and purified from a yeast culture, and thebiological activity compared to that of GIPFt that had been purifiedfrom the HEK 293 cell culture described above.

The nucleotide sequence encoding GIPFt (SEQ ID NO: 5) was cloned into aPichia expression vector pPICZαA which contains a yeast α-factorsecretion signal sequence. The Pichia Pastoris wild type X-33 strain wasused to express GIPFt. The protocols for the use of the Pichia vectors,expression and purification of recombinant proteins are available fromInvitrogen Life Technologies (Carlsbad, Calif., USA), and are alsodescribed in “Pichia Protocols: Methods in Molecular Biology” (D. R.Higgins and J. Cregg eds., The Humana Press, Totowa, N.J. 1998)).

Briefly, GIPFt was purified using the SP cation exchange chromatographyfollowed by affinity chromatography on an IMAC Ni²⁺ column. The Ni²⁺column was washed with 20 mM imidazole, and GIPF was eluted in a 20-300mM imidazole gradient over 30 column volumes. SDS-PAGE of the purifiedproduct displayed a broad and smeared protein band of about 50 kDa,indicating that GIPFt is glycosylated to varying degrees. The biologicalactivity analyzed in vitro and in vivo as described in Examples 17 and20, respectively.

GIPFt protein that was expressed in Pichia pastoris induced theproliferation of the mouse intestinal epithelium, and stabilizedβ-catenin, albeit to a lesser extent than that obtained with the GIPFprotein that was purified from HEK293 cells (data not shown).

D. Purification of the Murine Ortholog of GIPF-mGIPFt:

SEQ ID NO: 68, which encodes the mouse ortholog of the human GIPF, wascloned into the pcDNA/Intron vector to express a V5-His tagged proteinmGIPFt. The tagged mouse protein was expressed in HEK 293 cells, andpurified according to the method described above for the purification ofthe human GIPFt protein. The protein was purified to 80% purity, and wasformulated in PBS. The biological activity of mGIPFt was analyzed invivo and in vitro, and shown to possess the same proliferativeproperties as human GIPF (Example 20).

E. Characteristics of Purified Recombinant GIPF

SDS-PAGE analysis of the purified GIPF proteins (GIFPt and GIPFwt) wasperformed under reducing and non-reducing conditions, and showed thatthe V5-His tagged GIPF proteins derived from 293 cells exists as amonomer. GIPFt and GIPFwt proteins are glycosylated and migrate onSDS-PAGE under non-reducing conditions with a molecular weight (MW) ofapproximately 42 kDa and 38 kDa, respectively. Matrix-assisted laserdesorption/ionization mass spectroscopy (MALDI) showed that therespective molecular weights for GIPFt and GIPFwt are 37.8 kDa and 32.9kDa., while the theoretical molecular weight for GIPFt and GIPF wt thatlack the signal peptide is 30.2 kDa and 26.8 kDa, respectively. Thediscrepancy in the molecular weights suggested that it might have beenaccounted for by the glycosylation of the protein. Subsequently,complete deglycosylation of N-linked and O-linked oligosaccharides wasperformed using N- and O-glycanase (Prozyme, San Leandro, Calif., USA)according to the manufacturer's instructions. SDS-PAGE analysis of thedeglycosylated protein resulted in a decrease in apparent molecularweight of 4-5 kDa. Deglycosylation did not affect the biologicalactivity of GIPF when assayed in vitro and in vivo as described inExamples 17 and 20, respectively (data not shown).

Protein stability—To test the activity of GIPF following denaturation,GIPF was boiled for 5 minutes, and rapidly cooled on ice. GIPF retainedfull activity was determined in vitro (see Example 17) and in vivo (seeExample 21). These findings indicate that GIPF is a stable protein.

Capping of Cysteine residues—Reduction and alkylation of cysteineresidues was performed to abolish the activity of GIPF. Reduction of thedisulfide bonds of GIPF (1 mg/ml) was obtained by incubating GIPF in 30mM DTT, pH 8, at 37° C. for 1 hr. Subsequently, the free sulfhydrylswere by S-carboxymethylation with 20 mM Iodoacetamide at 37° C. in darkfor 30 minutes (Crestfield A M; Moore S; Stein W H. J. Biol. Chem. 1963;238, 622). The reaction was stopped by freezing, and excess DTT andIodoacetamide were removed by dialysis against PBS. The biologicalactivity of capped GIPF was analyzed using both in vitro and in vivoassays. The biological activity of GIPF was obliterated by capping (seeExamples 17 and 20).

N-terminal sequence analysis showed that HEK293 cells produced two formsof either the GIPFt or GIPFwt polypeptide: the dominant mature form (SEQID NO: 10) which corresponds to the GIPF protein of SEQ ID NO: 4 thatlacks the signal sequence, and the mature form (SEQ ID NO: 12), whichcorresponds to the GIPF protein of SEQ ID NO: 4 that lacks both thesignal peptide and the furin cleavage sequence. The two forms separatedwell on the SP column, and were expressed at a ratio of mature todominant mature forms of approximately 1:2. While both the dominantmature and mature forms of GIPFt induce proliferation of intestinalcrypt cells in vivo, the dominant mature form was used to test thetherapeutic effect of GIPF in the animal models of disease described inExamples 11, 12, 13, and 14. CHO cells express only the dominant matureform of GIPF.

Mutagenesis of Furin cleavage site (Arg²⁸->Gln)—To show that the matureform of GIPF produced from HEK 293 cells occurs from the naturalprocessing by furin protease, the conserved sequence of the furincleavage site was mutated to replace Arg²⁸QRR by Gln²⁸-QRR. The mutantprotein (SEQ ID NO: 18) was expressed in HEK293 cells and purifiedaccording to the method used for the purification of GIFPt (Example 9A).N-terminal sequencing of the purified protein confirmed that only thedominant mature form was expressed in the culture. This finding confirmsthat the mature form is generated as a result of proteolytic cleavage bycellular furin protease activity. In addition, the overall recovery ofthe purified dominant mature form increased from 50% to 68%.

In summary, the purification processes yield a GIPFt protein thatis >90% pure, and a GIPFwt that is 92-95% pure. The overall recovery ofthe dominant mature form of GIPF using either purification processes isapproximately 50%. However, the yield can be increased by expressing aprotein that has a mutated furin cleavage site. Addition of 0.5 M NaClto the buffer during the purification process of media diafiltration andNi column is crucial to keep GIPF fully soluble at pH 7. For bindingGIPF onto the SP column, NaCl was removed, and 0.3 M Arg was added tomaintain high solubility and increase protein recovery.

The dominant mature and mature forms of GIPFt and GIFP wt were used totest the biological activity of GIPF in vivo. The proteins purified bythe methods of this example consistently induced significantproliferation of intestinal crypt epithelial cells, which underlies thedistension of the small intestine of the mice that were administered thepurified GIPF protein.

The biological activity of GIPF was unaffected by deglycosylation orboiling, but was obliterated by capping cysteine residues withiodoacetamide.

Example 10 In Vivo Biological Testing of Recombinant GIPF ProteinExpressed In HEK293 and Cho Cells

The in vivo biological effects of GIPFt protein that was derived fromHEK293 and CHO cells were evaluated in normal mice as follows.

The pharmacokinetics (PK) of recombinant GIPF V5His6-tagged protein(GIPFt) were determined in mice. 6-8 weeks old BALB/c mice were injectedi.v. via the tail vein with single dose of either 40 mg/KG GIPFt proteinor formulation buffer as control. Blood was withdrawn at 0, 30 min, 1hr, 3 hr, 6 hr and 24 hr after injection and serum protein level at eachtime point was analyzed by Western analysis using anti V5 antibody(Invitrogene Inc., Carlsbad, Calif.) (FIG. 18A). FIG. 18A shows that nosignificant degradation of serum GIPF protein was detected. Thehalf-life of GIPF protein in serum was calculated by semi logarithmicplot of the protein concentration after injection using Positope(Invitrogene Inc., Carlsbad, Calif.) as a standard V5 tagged protein,and was estimated to be 5.3 hours (FIG. 18B).

To investigate whether purified recombinant GIPFt protein could generatea phenotype similar to that observed in the GIPF knock-in mice and inthe mice that had been injected with recombinant adenovirus, 6-8 weeksold BALB/c mice were injected daily through tail vein with either 4mg/KG GIPFt protein or formulation buffer as control for 7 days. Micewere sacrificed on day 8 at 24 hours after last injection. Four hoursprior to being sacrificed, 1 mg of bromodeoxyuridine (BrdU) was injectedip to determine the in vivo proliferative activity of GIPF. Varioustissues including small intestine, colon, spleen, liver and bone marrowwere collected and fixed in formaline. Paraffin embedded sections werestained with hematoxylin and eosin for histological evaluation. Sectionswere also processed for BrdU immunohistochemistry according to themanufacturer's instruction (Oncogene Research product, Boston, U.S.A.)and previously described (McKinley, J. N. et al. 2000). In allexperiments, at least 3 animals were analyzed per group and experimentswere repeated at least twice.

H&E staining of gastrointestinal sections showed that significantproliferation of intestinal crypt epithelial cells in the smallintestine and colon was seen in the mice that had received GIPF (FIGS.19 and 21, respectively). This result is consistent with the resultsobtained in the transgenic GIPF knock-in mice, and in the mice thatreceived GIPF adenovirus (see Examples above) The proliferative effectof GIPF protein was confirmed by assaying BrdU incorporation in bothsmall intestine (FIG. 20) and colon (FIG. 22).

Example 11 Prophylactic Effect of GIPF on Radiation-Induced Mucositis

The efficacy of GIPF as a prophylactic and therapeutic agent was testedin an animal model of radiation-induced mucositis.

Forty eight adult male BDF1 mice, aged 10-12 weeks, were used. Ondelivery from the supplier and prior to the experiment, the animals werehoused for two weeks in individually ventilated cages on a 12 hourlight:dark cycle to stabilize the circadian rhythm. Animals were allowedfood and water ad libitum.

The animals were divided into 8 groups of 6 animals, and were treated asfollows:1. Injected with 2 mg/kg GIPF iv at 72, 48, and 24 hours prior to beingexposed to 13 Gy X-ray (whole body);2. Injected with 5 mg/kg GIPF iv at 72, 48, and 24 hours prior to beingexposed to 13 Gy X-ray (whole body);3. Injected with 125 μg KGF iv at 72, 48, and 24 hours prior to beingexposed to 13 Gy X-ray (whole body);4. Injected with saline vehicle iv at 72, 48, and 24 hours prior tobeing exposed to 13 Gy X-ray (whole body);5. Untreated, non-irradiated controls;6. Injected with 2 mg/kg GIPF iv 24, 48, and 72 hours post irradiationwith 13 Gy X-rays (whole body);7. Injected with 5 mg/kg GIPF iv 24, 48, and 72 hours post irradiationwith 13 Gy X-rays (whole body);8. Injected with saline iv at 24, 48, and 72 hours post irradiation with13 Gy X-rays (whole body).

All injections were given at 15:00 hours. Intestinal damage was inducedusing a single dose of 13 Gy X-irradiation (delivered at 0.7 Gy/min) at15:00.

Four days after irradiation the animals were culled. The small intestinewas removed and fixed in Carnoy's fixative prior to processing forhistological analysis. Transverse sections 3 μm thick were cut andstained with haematoxylin and eosin. Immediately after sacrifice theduodenum, mid colon, liver, lung, tongue, spleen, stomach and pancreaswere also removed and fixed in formal saline overnight prior to storagein 70% ethanol.

For each animal ten intestinal circumferences were analyzed (60 pergroup)—a circumference is equivalent to a given length of intestine andtherefore a convenient baseline unit of length. The number of survivingcrypts per circumference were scored and the average per groupdetermined (Table 4). Only crypts containing 10 or more strongly H&Estained cells (excluding Paneth cells) and only intact circumferencesnot containing Peyers patches were scored.

The average crypt width (measured at its widest point) was also measuredin order to correct for scoring errors due to crypt size difference. Thecorrection is applied thus:

$\frac{{Corrected}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {crypts}}{circumference} = {\frac{{Mean}\mspace{14mu} {crypt}\mspace{14mu} {width}\mspace{14mu} {in}\mspace{14mu} {untreated}\mspace{14mu} {control}}{{Mean}\mspace{14mu} {crypt}\mspace{14mu} {width}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {animal}} \times {Mean}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {surviving}\mspace{14mu} {crypts}\mspace{14mu} {in}\mspace{14mu} {treatment}\mspace{14mu} {group}}$

All the animals survived the treatment and exhibited no obvious adverseeffects. FIG. 23 A-D shows sections from the small intestine from theanimals the untreated group 5 (A), the saline pre-treated group 4 (B),the KGF-treated group 3 (C) and the GIPF-treated group 2 (D). Foci ofregeneration (surviving crypts with one or more clonogenic cells) areclearly visible in the tissue section from the saline-treated animals(group 4) (FIG. 23B) Other than these foci the mesenchyme is entirelydenuded, and these animals would have developed diarrhea and died due tothe mucositis if they had been allowed to live beyond four days postirradiation. GIPF afforded protection of the intestinal architecture(FIG. 23 D) in a manner comparable to the to that provided by KGF (FIG.23 C.).

TABLE 4 No. of crypts/ Crypt Width Corrected crypts/ ExperimentalCircumference (μm) Circumference Group (Mean ± SD) (Mean ± SD) (Mean ±SD) 1. 12.96 ± 4.9  51.98 ± 5.4 7.4 ± 3.3 2. 15.4 ± 4.9 45.03 ± 2.8 10.1± 3.3  3. 22.1 ± 3.8 54.46 ± 2.1 12.0 ± 1.2  4.  7.2 ± 2.6 55.27 ± 6.43.8 ± 1.2 5. 109.1 ± 5.3  29.56 ± 3.0 6. 10.7 ± 4.5 57.16 ± 3.9 5.5 ±2.3 7. 10.6 ± 4.1 55.50 ± 4.7 5.6 ± 2.1 8.  9.4 ± 1.9 56.85 ± 6.9 5.0 ±1.4

It is immediately obvious that pre-treatment with GIPF increasedconsiderably the number of crypts that survived the 13 Gy irradiation.Pre-treatment with GIPF at a dose of 2 mg/kg (group 1) increasedsurvival by 1.95 fold that of the crypts from the untreated group 4(also known as the protection factor), and the GIPF dose of 5 mg/kg(group 2) further increased crypt survival by 2.66 fold. This isextremely impressive and almost comparable with the effect seen withtreatment with the known optimal dosing of KGF (group 3), whichincreased crypt survival by 3.16 fold.

Therefore, GIPF was shown to protect the epithelium of the smallintestine from the injurious effects of irradiation, and could be usedas a potent prophylactic in patients for whom radiation therapy has beenindicated.

Example 12 Chemotherapy-Induced Mucositis Model

The efficacy of the recombinant human GIPF (GIPFwt) in treatingchemotherapy-induced mucositis was evaluated in healthy and intumor-bearing mice. The experimental protocol was based on thatpreviously described by Boushey et al. (Cancer Res 61:687-693 (2001)).

One million CT26 murine colon carcinoma cells (ATCC, Manassas, Va., USA)were injected sc into syngeneic female BALB/c mice, and the tumors wereallowed to develop for 5 days. Healthy and tumor-bearing animals weredivided into experimental groups of 6 mice each and treated as follows:

1. tumor bearing mice, vehicle (50% DMSO) injected ip from day 1 to day5, saline injected iv from day 0 to day 7 (TVS)2. tumor bearing mice, vehicle (50% DMSO) injected ip from day 1 to day5, 50 μg GIPF in 100 μl saline injected iv daily from day 0 to day 7(TVG);3. tumor bearing mice, 50 mg/kg 5-FU injected ip from day 1 to day 5,saline injected iv from day 0 to day 7 (TDS);4. tumor bearing mice, 50 mg/kg 5-FU injected ip from day 1 to day 5, 50μg GIPF in 100 μl saline injected iv from day 0 to day 7 (TDG);5. healthy mice, 50 mg/kg 5-FU injected ip from day 1 to day 5, salineinjected iv from day 0 to day 7 (NDS);6. healthy mice, 50 mg/kg 5-FU injected ip from day 1 to day 5, 50 μgGIPF in 100 μl saline injected iv from day 0 to day 7 (NDG).

On days 0, 2, 4, 6, and 8 measurements of animal body weight, severityof diarrhea, and size of the tumors were recorded. A diarrhea score of0-3 reflected a corresponding worsening of the symptom from 0 beingnormal to 3 being severe. The change in body weight was calculated asthe percent body weight of that of the untreated group. The length,width and height of the tumor were measured with calipers, and thevolume of the tumor was calculated as (length×width×height)/2.

All animals were euthanized on day 8. The large and small intestine wereremoved and weighed, their length was measured, and the diameter of themid-jejenum was recorded. A segment (1 cm) of the mid-jejenum wasexcised about 14-15 cm from the pylorus, and a segment (1 cm) of thetransverse colon was excised at about 4 cm from the ileocaecal junction.The bowel segments were flushed and fixed using 10% neutral bufferedformalin for histological analysis. Histological examination andmorphometry of the mucosa were performed on tissue sections using theImagePro Software (Imagepro, Ltd., Ashford, Middlesex, UK).

The effect of GIPFwt on the severity of diarrhea, body weight and tumorsize are summarized as follows:

Diarrhea score (mean ± SD): Body weight (% of untreated) Group 3 TDS2.83 ± 0.41 74.7 ± 5.2 Group 4 TDG 0.33 ± 0.52 85.1 ± 5.7 Group 5 NDS 3± 0 73.2 ± 4.3 Group 6 NDG  0.5 ± 0.55 87.0 ± 6.0 Tumor volume (mean ±SD; mm³): Group 1 TVS 95.8 ± 8.1 Group 2 TVG 95.1 ± 4.2 Group 3 TDS 21.8± 3.0; p < 0.05 Group 4 TDG 16.7 ± 8.6; p < 0.05

GIPF reduced significantly the severity of the diarrhea caused by 5-FUin the healthy and the tumor-bearing mice of groups 6 and 4,respectively, when compared to the scores for the normal andtumor-bearing mice of groups 5 and 3, which did not receive GIPF.Similarly, GIPF reduced the loss of body weight that the 5-FU-treatedanimals experienced.

The tumors from the untreated tumor-bearing mice (group 1) were similarin size to those from the GIPF-treated tumor-bearing mice (group 2).Thus, GIPF did not affect the growth of the tumor. As expected, 5-FUreduced the size of the tumors in the mice of group 3, and it alsoreduced the size of the tumors of the mice of group 4 Thus, GIPF did notimpede the activity of 5-FU in reducing the size of the tumors (FIG.24).

The effect of GIPF on the gross appearance of the intestines is shown inFIG. 25, and the corresponding measurement of intestinal diameter,weight and length are given in Table 5. The intestines of the normal andtumor-bearing mice that had received 5-FU was atrophied (FIG. 25E), andnumerous lesions associated with bleeding were observed, while theappearance of the intestines from the mice that had received GIPF wasovertly normal and accompanied by the typical distension due to theproliferative effect of GIPF on the in intestinal epithelium (FIGS. 25B, C, D, and F).

TABLE 5 CT26 tumor-bearing mice Group 1 Group 2 Group 3 Group 4 Diameter2.66 ± 0.15 3.65 ± 0.21* 2.53 ± 0.15 3.58 ± 0.14^(#) Midjejenum (mm)Weight Small bowel 1.16 ± 0.09 1.47 ± 0.14* 0.90 ± 0.01 1.51 ± 0.13^(#)(g) Large bowel 0.32 ± 0.02 0.38 ± 0.02* 0.25 ± 0.03 0.39 ± 0.02^(#) (g)Length Small bowel 35.2 ± 2.0  40.5 ± 1.0*  30.2 ± 1.3  39.7 ± 2.1^(#) (cm) Large bowel 8.7 ± 0.3 9.7 ± 0.3* 6.3 ± 0.3 9.5 ± 0.5^(#) (cm)Normal mice Group 5 Group 6 Diameter 2.38 ± 0.13 3.44 ± 0.13**Midjejenum (mm) Weight Small bowel (g) 0.88 ± 0.08 1.43 ± 0.13** Largebowel (g) 0.25 ± 0.02 0.38 ± 0.04** Length Small bowel (cm) 30.3 ± 1.9 39.3 ± 1.0**  Large bowel (cm) 6.9 ± 0.9 9.7 ± 1.0** *P < 0.05 (ANOVA,group 2 vs group 1) ^(#)P < 0.05 (ANOVA, group4 vs group 3) **P < 0.05(ANOVA, group 6 vs group 4)

Histological analysis of intestinal sections of the small intestine andcolon of all experimental groups showed that GIPF preserved theintestinal architecture of the mice that had received 5-FU by preventingthe massive damage to the villi and crypt compartments of the intestinalmucosa caused by 5-FU (FIG. 26), FIG. 26 A shows the effects of 5-FU onthe small intestine, and FIG. 26B shows the effects of 5-FU on thecolon. Micromorphometry measurements of villus height and crypt depth inthe midjejenum confirm that the effect of GIPF is significant (FIG. 27),

GIPF protects that small intestine and colon from the deleteriouseffects of 5-FU, and it does not hinder the therapeutic effects of 5-FU.Therefore, GIPF may be used in conjunction with chemotherapeutic agentsto reduce the deleterious side-effects of antineoplastic therapies.

Example 13 Prophylactic Effect of GIPF on Chemotherapy andRadiation-Induced Oral Mucositis

The effect of GIPF on the proliferation of the dorsal (buccal) andventral epithelium of the tongue was studied in mice that had beensubjected to X-ray irradiation or dosed with 5-FU, as described inExamples 11 and 12, respectively.

Immunohistochemistry using monoclonal rat anti-mouse Ki67 antigen (DakoLtd., High Wycombe, UK) was performed, according to manufacturer'sinstruction and the method previously described (Scholzen, T. et al.2000), on paraffin embedded sections of tongue from non-irradiated andirradiated mice (groups 1, 2, and 3 in Example 11).

GIPF visibly increased the number of nuclei that stained for Ki67 in theventral and dorsal tongue epithelium of irradiated animals when comparedto that from animals that were not given GIPF (FIGS. 28 and 29). Theepithelial proliferative index, which is calculated as the percentventral epithelial cells that stained positive for Ki67, confirmed thatGIPF reduced significantly the loss of cellularity caused by theradiation to the ventral tongue epithelium (FIGS. 30).

Histological analysis of sections from the tongue of animals that hadbeen treated with 5-FU (groups 2-6 in Example 12) shows that GIPFmaintains the morphology of the dorsal and ventral epithelial layers innormal and tumor-bearing animals that had been treated with 5-FU (FIG.31).

The epithelial layer of the tongue from all animals that had beentreated with GIPF was remarkably less damaged by 5-FU than that of theexperimental animals that had not received GIPF.

Therefore, GIPF may be used as a therapeutic agent for the treatmentand/or prevention of chemotherapy and radiation therapy-induced oralmucositis. Quantitative animal models of oral mucositis (e.g. Wardly etal., Arch Oral Biol 43:567-577 (1998); Potten et al., Cell Prolif35:32-47 (2002)) can be used to study further the therapeutic propertiesof GIPF, when administered in combination with other cytotoxic agent tofurther assess the potential role of GIPF in reducing the severity ofthe cellular depletion and to increase the rate of regeneration of theepithelial layers of the oral and intestinal epithelium.

Example 14 Therapeutic Effect of GIPF on Dextran Sulfate Sodium-InducedColitis

The efficacy of recombinant human GIPF (GIPFwt) in treating colitis wastested in a mouse model of dextran sulfate sodium (DSS)-induced colitis,and compared to that the efficacy of GLP-2 (L'Heureux and Brubaker JPharmacol Exp Ther 306:347-354 (2003); Kriegelstein et al., J ClinInvest 110:1773-1782 (2002); Siegmund et al., J Pharmacol Exp Ther296:99-105 (2001)).

Six to eight-week old female BALB/c mice (Charles River Laboratories,Wilmington, Mass., USA) were housed in ventilated cages and acclimatedfor one week to a 12 hour light:dark cycle. Twenty four mice havingsimilar body weight (approximately 20 g; <5% variance) were housed in 4cages and fed ad libitum a 4% DSS (v/w) drinking solution for 7 days.

On day 7, the body weight of each animal was recorded, and the scoresfor loss in body weight, the consistency of stools, and anal bleedingwere determined as shown in Table 6 below.

TABLE 6 Occult/Gross Rectal SCORE Weight Loss (%) Stool Consistencybleeding 0 None Normal Normal 1 0-5% 2  5-10% Loose Hemoccult 3 10-20%4 >20% Diarrhea Gross

The scores were used to calculate the IBD activity index (IBDAI), whichwas used as an indicator of the severity of the colitis, and iscalculated as the average of the scores given for the tabulatedparameters. The scores for weight loss, stool consistency, and rectalbleeding were determined daily, and the IBDAI was recorded daily for theduration of the experiment.

On day 7, the 4% (v/w) DSS drinking solution was substituted with a 1%(v/w) DSS solution to maintain the disease activity without exacerbatingthe effect of the DSS. Sixteen of the DSS-fed animals were selected forconsistent and comparable disease activity, and were dived into groupsof 4 animals and were treated as follows:

-   -   1. Water, saline injected iv daily (10 am) for 7 days    -   2. DSS (1%) for 7 days, saline injected iv daily (10 am) for 7        days    -   3. DSS (1%) for 7 days, 100 μg GIPF injected daily iv (10 am)        for 7 days    -   4. DSS (1%) for 7 days, 50 μg GIPF injected daily iv (10 am) for        7 days    -   5. DSS (1%) for 7 days, 10 μg GLP-2 injected sc twice daily (10        am and 6 pm) for 7 days.

The GIPF protein used in these experiments was the human recombinantGIPF protein (SEQ ID NO: 4; GIPFwt), which was expressed and purifiedaccording to the method described in Example 9. The analog of GLP-2,h[Gly²]GLP-2 was synthesized and purchased from Biosource International(Camarillo, Calif., USA).

On day 14, food was removed from the cages to allow for purging of theintestine, and the animals were culled by cervical dislocation. Allanimals were injected with 4 mg/0.1 ml BrdU two hours prior tosacrifice. The large and small intestine were removed and weighed, theirlength was measured, and the diameter of the mid-jejenum was recorded. Asegment (1 cm) of the mid-jejenum was excised about 14-15 cm from thepylorus, and a segment (1 cm) of the transverse colon was excised atabout 4 cm from the ileocaecal junction. The bowel segments were flushedand fixed using 10% neutral buffered formalin for histological analysis.Histological examination and morphometry of the mucosa were performed ontissue sections using the Imagepro Software (Imagepro, Ltd., Ashford,Middlesex, UK). The IBDIAs for the mice of experimental groups 2-5 areshown in FIG. 32, and the corresponding scores for weight loss, stoolconsistency and rectal bleeding are shown in FIGS. 33, 34, and 35,respectively. These data show that GIPF afforded a therapeutic effect byreducing the severity of the colitis as early as day 11. By day 14, theGIPF-treated mice of groups 3 and 4 had significantly lower IBDIA (1.75and 1.83, respectively) than the untreated mice of group 2 (3.75). GLP-2had a moderate effect on the severity of the colitis, and by day 14 itreduced the IBDAI of the DSS-treated mice to 3.25.

An example of the gross pathology of the intestine and colon of the micefrom groups 1, 2, 3, and 5 is shown in FIG. 36. Animals receiving DSSwith saline developed severe colitis that was typically associated withatrophy, hyperemia, and diarrhea when compared to the control group. Thesmall and large intestine of the animals that were treated with GIPFshowed some distension, and were remarkably similar to those of thecontrol group. These findings indicate that GIPF may be used as aneffective therapeutic agent for the treatment of inflammatory boweldisease. While GLP-2 afforded some therapeutic effect, the small andlarge intestine from this group seemed marginally less injured than thatfrom the animals in the control group. The significance of the changesis reflected by the measurements of the small and large intestines shownbelow in Table 7.

TABLE 7 Group 1 Group 2 Group 3 Group 4 Group 5 Diameter Midjejenum (mm)2.25 ± 0.09 1.72 ± 0.05* 2.50 ± 0.18^(#) 2.23 ± 0.10# 1.97 ± 0.04**Weight Small bowel (g) 0.94 ± 0.06 0.78 ± 0.05* 0.91 ± 0.09^(#) 0.89 ±0.05^(#) 0.88 ± 0.07 Large bowel (g) 0.26 ± 0.02 0.18 ± 0.01* 0.23 ±0.01^(#) 0.22 ± 0.01^(#) 0.18 ± 0.01 Length Small bowel (cm) 32.0 ± 1.426.6 ± 0.9* 31.1 ± 0.9^(#) 30.3 ± 0.6^(#) 28.9 ± 0.8** Large bowel (cm) 7.3 ± 0.3  4.9 ± 0.5*  6.6 ± 0.5^(#)  6.4 ± 0.3^(#)  5.4 ± 0.6 *P <0.05 (ANOVA, group 2 vs group 1) ^(#)P < 0.05 (ANOVA, groups 3 or 4 vsgroup 2) **P < 0.05 (ANOVA, group 5 vs group 2)

H & E staining of paraffin embedded sections showed that DSS causedmassive infiltration by inflammatory cells and disintegration of thevillus and crypt compartments of the mucosa of the small intestine andcolon (FIG. 37). Consistent with the observations of the gross pathologydescribed above, GIPF reversed the effects caused by DSS, and restoredthe intestinal architecture of the crypts and villi. The crypts of theGIPF-treated animals were distended when compared to those from thecontrol group. GLP-2 afforded some therapeutic effect, albeit to a farlesser level than GIPF (data not shown). Micromorphometry of the villiand crypts (FIG. 38) confirmed that the curative effect of GIPF wasreflected by a significant restoration of the villus height and thecrypt depth, which had been severely destroyed by the colitis. Therepair of the mucosal architecture by GIPF was underscored by asignificant proliferation of crypt cells (FIGS. 39 and 40). The cryptproliferative index, which is calculated as the percent crypt cells thatstained positive for BrdU, was significantly greater in the DSS-treatedmice that received GIPFwt than in the DSS-treated mice that wereinjected with saline (P<0.05) (FIG. 40).

Example 15 Therapeutic Effect of GIPF Following Massive IntestinalResection

The effect of GIPF in augmenting the adaptive response to massiveintestinal resectionis tested in a rat animal model of short bowelsyndrome (SBS). The animal model used in the study of the effects ofenterorophic agents has been described (Scott et al. Am J PhysiolG911-G921 (1998); Helmrath et al., J Am Coll Surg 183:441-449 (1996)),and the experimental protocol is herein incorporated by reference).

The animals are divided into a resected group that will have a 75%surgical resection of the midjejenunoileum, a sham-resected operatedcontrol group in which the intestine is sectioned and reanastomosed, andan unoperated control group. The animals are administered saline or GIPFat a dose of 2 mg/Kg. The 75% intestinal resection is chosen to maximizeany adaptive response, and retention of equal portions of the proximaljejunumnum and distal ileum is based on the nutritional implications ofremoving the specialized absorptive capacity of the terminal ileum forvitamin B12 and bile acids and the ileal brake. In the rat, theretention of 25% of the small intestine inclusive of a portion of distalileum, is sufficient to allow resected animals to achieve the samegrowth rate as control animals.

The metabolic, morphological, histological, and functional response ofthe gut to resection and treatment with GIPF is assessed during thecourse of the experiment and also as end-point analysis on Day 10. Foodintake and growth, gross and microscopic small intestinal morphology,and functional evaluation of mucosal absorptive characteristics areevaluated as described (Scott et al., supra).

GIPF significantly increases the food consumption and reduces the lossin body weight that typically accompanies resection of the small bowel.GIPF also increased the length of the remnant intestine, its diameter,wet weight, and mucosal wet weight, and increases the absorptivecapacity of the remnant small intestine. H&E staining of cross-sectionsof the small intestine shows that GIPF elongates both the villus heightand crypt depth, and increases crypt cell proliferation in the gut ofthe animals with resected small intestines. Thus, GIPF reduces theeffects of bowel resection by augmenting intestinal adaptation.

Example 16 Effect of GIPF on the Proliferation of Tumor Cells

GIPF induces a strong proliferative effect on intestinal cryptepithelial cells in vivo. To investigate the proliferative effect on invitro, the effect of recombinant GIPFwt was tested on the proliferationof various tumor and normal cell lines in vitro.

The rate of cell proliferation of the following cell lines (ATCC) wasmeasured by assaying the incorporation of ³H-thymidine:

Caco-2 human colorectal adenocarcinoma; epithelial COLO205 human ascitesfrom metastatic colorectal adenocarcinoma; epithelial HCC70 humanmammary gland ductal carcinoma; epithelial HCT116 Human colorectalcarcinoma; epithelial HT-29 Human colorectal adenocarcinoma; epithelialIEC-18 Rat ileum; epithelial IEC-6 rat small intestine; epithelial LS513human caecum; colorectal carcinoma; epithelial MCF7 human pleuraleffusion from metastatic breast adenocarcinoma; epithelial NCI-H1373human lung adenocarcinoma PC-3 human bone metastasis from prostateadenocarcinoma; epithelial SCC-25 Human tongue squamous cell carcinomaSCC-4 Human tongue squamous cell carcinoma SK-BR-3 Human pleuraleffusion from metastatic colon adenocarcinoma; epithelial SK-MES-1 Humanpleural effusion from metastatic squamous cell lung carcinoma;epithelial SW620 Human lymph node metastasis from colorectaladenocarcinoma; epithelial T84 Human lung metastasis from colorectalcarcinoma; epithelial 293 Human fetal kidney; epithelialCells were seeded at 10,000-50,000 cells per well, and depending on celllines and treated with scaled doses of GIPFwt (1.37-1000 ng/ml). Therate of proliferation of the GIPF-treated cells was compared to that ofuntreated cells, or cells that were grown in 10% complete media (growthmedia, 2.5% dialyzed FBS, and pen/strep). The cells were incubated for48 hours at 37° C., and pulsed with 0.5 μCi ³H-thymidine for the last20-24 hours of incubation. Cells were harvested, the amount of³H-thymidine that had been incorporated was determined, and the resultsdetermined from duplicate samples of replicate experiments.

GIPFwt did not affect the rate of proliferation of most of the tumorcells that were tested. An increase in the rate of proliferation wasinduced only at the higher doses of GIPF in IEC28, T84, HCT116, and HT29cells. The extent of the proliferation was less than 40% of rate of theuntreated cells.

Therefore, these findings indicate that GIPF may not exacerbate the rateof proliferation of tumors existing in vivo, and GIPF may be used fortreating cancer patients who are suffering from mucositis caused byantineoplastic therapies.

Example 17 Effect of GIPF on Intracellular Signaling

The wnt/β-catenin signaling pathway plays a pivotal role in developmentand homeostasis. In the small intestine, wnt signaling is known to playa critical role as a regulator of intestinal crypt proliferation bystabilizing β-catenin, which subsequently induces the transactivation ofT-cell factor (TCF) target genes (Wetering et al., Cell 111:241-250(2002); Batle et al., Cell, 111:251-263, (2002); Perreault et al., JBiol Chem 276:43328-43333 (2001); Booth et al., Nat Med 8:1360-1361(2002)).

To evaluate the effect of GIPF on the wnt/β-catenin signaling pathway,the stabilization of β-catenin was measured in various cultured celllines. Cells were seeded at 1 million cells/well for a 6-well plate inDulbecco's modified Eagle's medium supplemented with 10% FBS. Thefollowing day, cells were grown in serum-free medium, and treated eitherwith GIPF at 50 ng/ml or LiCl₂ (positive control) at 10 mM in low serumconditions (0.1% FBS). Cytoplasmic fractions were prepared as describedby Haertel-Weismann et al., (J Biol Chem 175:32046-32051 (2000)). Theproteins were resolved by gradient (4-20%) SDS-PAGE, and the level ofβ-catenin was assessed using a β-catenin rabbit antibody (Abcam) thatwas visualized using a peroxidase conjugated secondary antibody (CellSignaling).

Among tested cell lines, GIPF induced the stabilization of β-catenin ina human endocrinic L cell line (NCI-H716, data not shown), and in HEK293 cells in a dose-dependent and time-related manner (FIGS. 41A and B,respectively). Consistent with the findings described in Example 18,boiling GIPF did not affect its ability to stabilize β-catenin, but, theeffect was abolished by treatment with proteinase K, and by reductionwith DTT (FIG. 41 C).

To further investigate the signaling pathway through which GIPF leads tothe accumulation of β-catenin, the activity of GSK3β was analyzed inHEK293 cells. In the canonical wnt signaling pathway, Wnt activatesβ-catenin by inhibiting GSK3β, which would otherwise phosphorylateβ-catenin and mark it for destruction by the proteosome.

GIPF increased the phosphorylation of GSK3β in HEK 293 cells in atime-dependent manner (FIG. 42). These data indicated that GIPF mayactivate β-catenin by canonical wnt signaling pathway by inhibitingβ-catenin phosphorylation by GSK3. However, GIPF did not induce thestabilization of p-catenin in other cell lines including the mouseepithelial cell line C57MG in which Wnt3A has been shown to have apotent effect on induces β-catenin activation. Furthermore, Dickkopf-1(Dkk1), which is a potent inhibitor of the Wnt signaling pathway(Kuhnert, PNAS 101:266-271, 2004), did not completely inhibit β-cateninstabilization by GIPF in 293 cells (data not shown). These data suggestthat GIPF may stabilize β-catenin via a pathway that is distinct fromthe known canonical Wnt/β-catenin pathway.

Example 18 Effect of GIPF on the Expression of β-Catenin Target Genes

Accumulation β-catenin results in its translocation to the nucleus whereit associates with transcription factors of the TCF/LEF family. Due toits transactivating ability the β-catenin-transcription-factor-complexbinds to DNA and activates wnt target genes. To further investigateGIPF-induced β-catenin signaling, we determined the activation of downstream target genes in HEK293 and NCI-H716 cells by quantitative PCR.

1×10⁶ HEK-293 cells and 2×10⁶ NCI-H716 cells (ATCC) were seeded in6-well plates and allowed to attach 6 hrs in complete media. Cells werethen changed to 0.1% FBS Assay media and incubated overnight. The day ofthe assay, treatments were added to the cells in an additional 1 ml ofAssay media. Cells were incubated for 8 hours at 37° C./5% CO2 witheither 20 mMLiCl (Sigma), 10 ng/ml Wnt-3A (R&D Systems), 250 ng/mlGIPFt, or 250 ng/ml capped GIPF protein. A well of untreated cells,maintained in Assay media, was included as a background for geneexpression. Total RNA was isolated from both cell types using RNeasyMini kit and DNasel kit (Qiagen), quality and concentration of the totalRNA was quantified. For each sample, 4 ug of total RNA was primed at 70°C. for 3 mins with 3 ug Random Hexamers and 2 mM each dNTP. Reactionswere cooled on ice for 1 min. Reaction volume was brought to 22 ul with5×M-MLV Buffer (Promega), 25 mM MgCl₂, 0.1 M DTT and RNaseOut(Invitrogen). Upon addition of 400 units M-MLV Reverse Transcriptase(Promega) the reactions were incubated 10 mins at 23° C., 50 mins at 42°C., and 5 mins at 70° C. to terminate the reaction. cDNA was thendiluted and treated with 1 unit of RNaseH (Invitrogen) to digestremaining RNA. OD260 nm quantified cDNA concentration. For each 10 ulquantitative SYBR Green PCR reaction, 2 ul cDNA (440 ng) was used, inconjunction with 1.25 μM of each forward and reverse primer, and 2×SYBRGreen mastermix (Eurogentec). Reactions were performed in triplicate.Primers for Quantitative PCR were designed for the following humanβ-catenin target genes: Axin-2 (SEQ ID NOs: 70 and 71), CD44 (SEQ ID NOs72 and 73), EpherinB2 (SEQ ID NOs: 74 and 75), c-myc (SEQ ID NOs: 76 and77), Proglucagon (SEQ ID NOs: 78 and 79), and Cox-2 (SEQ ID NOs: 80 and81). Human EF1 (SEQ ID NOs: 82 and 83) was used as housekeeping gene tostandardize expression levels.

GIPF increased the expression of Axin-2 in HEK-293 and NCI-H716 cells,and caused the upregulation of CD44 and EphrinB2 to levels that weregreater than resulting from stimulation with Wnt3A or Lithium. Theexpression levels of Cox-2, c-myc and proglucagon genes were notaffected by GIPF (data not shown).

These data provided insight into the mechanisms involved in GIPF-inducedtarget gene activation. Further studies are performed to elucidate theevents downstream of GIPF signaling.

Example 19 In Vitro Assay for the Activity of GIFP

Eleven deletion mutants of GIPF (SEQ ID NOs: 84, 86, 88, 90, 92, 94, 96,98, 100, 102, and 104) were subcloned into the pIntron/IgK vector, andtransiently expressed in HEK293 cells. The position of each of theencoded polypeptide fragments (SEQ ID NOs: 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105 and 178) within the full-length GIPF polypeptide isshown in FIG. 43. The mammalian expression vector pIntron/IgK wasobtained by genetically modifying the pSectag vector (Invitrogene Inc.,Carlsbad, Calif.) by introducing an engineered chimeric intron derivedfrom the pCI mammalian expression vector (Promega, Madison, Wis.).pcDNA/Intron vector was digested with BGIII and NheI, and the intronsequence was cloned into pSectag, which had been digested with BgIII andNheI. The forward and reverse primers used to amplify and subclone thepolynucleotide fragments correspond to SEQ ID NOs: 106-119, as indicatedin the sequence listing. The forward primer of SEQ ID NO: 106 was usedwith the reverse primers for fragments 1-7 (primer SEQ ID NOs: 107-113;the forward primer of SEQ ID NO: 114 was used with the reverse primersfor fragments 8-10 (SEQ ID NOs: 115-117).

The polypeptide fragments were transiently expressed in HEK 293 cellsand the activity of the fragments in stabilizing β-catenin was assayedas described in Example 17. The polypeptide fragment of SEQ ID NO: 91induced the greatest stabilization of β-catenin when compared to theactivity displayed by the other fragments tested. This finding suggeststhat the furin-like cysteine-rich domain of GIPF may be essential formediating the proliferative activity of GIPF. However, the activity ofthe polypeptide of SEQ ID NO: 91 was lower than that of the full-lengthGIPF (FIG. 44). Therefore, other portions of the GIPF protein arenecessary to enable the maximum stabilization of β-catenin.

Example 20 Rapid In Vivo Assay for the Activity of Recombinant GIFP

A rapid in vivo assay was developed to test the test the bioactivity ofpurified GIPF.

The activity of human and mouse GIPF (GIPFwt and mGIPFt) was tested inmice that had been grouped and treated as follows:

1. Saline, injected i.v.

2. GIPFwt, injected 100 μg i.v.

3. GIPFwt, injected 50 μg i.v.

4. mGIPFt, injected 100 μg i.v.

5. mGIPFt, injected 50 μg i.v.

6. GIPFwt, boiled, injected 100 μg i.v.

7. GIPFwt, capped, injected 100 μg i.v.

8. GIPFwt, injected 100 μg s.c.

Twenty four female BALB/c mice were used in the experiments. GIPF wasinjected daily for three days. Animals were sacrificed on day 4. Priorto being sacrificed, 0.5 ml of blood was collected for hematologicalanalysis, and two hours prior to being sacrificed, all animals wereinjected i.p with 0.4 ml of a 1 mg/ml solution of BrdU. The smallintestine and colon were dissected, measured as described above, andsegments of midjejenum and colon processed for histological analysis asdescribed in previous Examples.

The results are shown in FIG. 45. The distension of the intestines fromthe mice that had received mouse GIPF protein, mGIPFt, was comparable tothat induced by the human GIPFwt. In addition, the phenotype of theintestines of the mice that received GIPFwt via subcutaneousadministration was comparable to that of the mice that had receivedGIPFwt via the i.v. route. As previously noted, boiling of GIPFwt doesnot affect its ability to cause the distension of the intestine. Thesefindings were consistent with the measurements of length, weight, anddiameter of the intestines from all animal groups (Table 8).

TABLE 8 Diameter (mm) Weight (g) Weight (g) Length (cm) Length (cm)midjejenum Small bowel Large bowel Small bowel Large bowel Animal GroupMean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD 1 2.31 ± 0.15 0.90 ±0.04 0.24 ± 0.03 29.5 ± 1.5 6.7 ± 0.3 2 3.64 ± 0.14* 1.34 ± 0.10* 0.33 ±0.02* 36.0 ± 1.3* 8.5 ± 0.5* 3 3.55 ± 0.12* 1.32 ± 0.07* 0.32 ± 0.03*36.5 ± 0.5* 8.7 ± 0.3* 4 3.68 ± 0.06* 1.39 ± 0.05* 0.35 ± 0.02* 37.8 ±0.8* 8.8 ± 0.3* 5 3.45 ± 0.09* 1.23 ± 0.04* 0.33 ± 0.01* 36.5 ± 0.5* 8.5± 0.5* 6 3.07 ± 0.12* 1.25 ± 0.06* 0.34 ± 0.01* 35.5 ± 0.5* 8.3 ± 0.3* 72.28 ± 0.11 0.88 ± 0.07 0.21 ± 0.03 31.0 ± 1.3 6.3 ± 0.3 8 3.03 ± 0.10*1.12 ± 0.04* 0.28 ± 0.02* 34.2 ± 0.8* 7.8 ± 0.3* *P < 0.05 (ANOVA,groups 1-8 vs group 1)

All hematological results were within the normal range for the animals,thus indicating that GIFP did not produce any immediate adverse effects.The effect of GIPFwt and mGIPFt on villus height and crypt depth showedthat GIPF significantly increased crypt depth in healthy mice after the3-day regimen. In contrast to the effect of GIPF on increasing thevillus height in the intestine of diseased animals, GIPF did not affectthe height of the villi from normal, healthy animals. (FIG. 46).

These data show that the proliferative effect of the human recombinantGIPF is not the result of an ectopic effect. In addition, GIPF exhibitsits biological activity whether it is administered intravenously ofsubcutaneously.

Example 21 In Vitro Assays for the Activity of GIPF in Isolated CryptCells

The effect of GIPF on signaling and proliferation of crypt epithelialcells, was assayed in isolated mouse colonic crypt cells according tothe method of Fujimoteo, et al., (Fujimoto et al., Gastroenterology117:858-865 (2002)).

Colons were dissected from mice and sterilized in a 0.04% sodiumhypochlorite solution for 15 minutes. After rinsing in PBS, colons wereincubated in the DTT/EDTA solution (0.5 mM DTT, 3 mM EDTA in PBS) for 90min at room temperature. After the incubation, the tissue was washedonce in PBS and 10 ml of PBS was added. The tube was shaken vigorouslyto liberate the crypts from the submucosa. The PBS containing the cryptswas transferred to a centrifuge tube and the shaking step was repeateduntil the crypt yield diminished. The crypts were centrifuged gently(400 rpm for 5 min) and washed with fresh PBS. The crypts wereresuspended in 20 ml of 0.3% pancreatin (Sigma) in PBS and incubated for90 min at room temperature shaking every 10 minutes for first 30 minutesand every 30 minutes there after. At the end of the incubation, an equalvolume of PBS was added and the crypts were centrifuged at 1000 rpm for5 min and washed with EDTA/DTT solution 1-2 more times until all mucouswas removed. Crypt cells were resuspended in 1× media (RPMI 1640supplemented with 5% FCS, glutamine, NaHCO₃, insulin, transferrine,selenium, penicillin/streptomycin). Cell clumps were broken up using 21Gthen 23G needles with syringe. Cells were counted and used to assay forthe stabilization of β-catenin; for determining the proliferativeactivity by incorporation of ³H-thymidine; and for testing the abilityof GIPF to affect the clonogenicity of crypt cells.

A. The effect of GIPF on the activation of p-catenin in vivo was studiedin crypt cells that had been isolated from Balb/c mice 6 hours afterthey had been injected i.v. with 100 μg of GIPFwt, as described above.

As shown in FIG. 47, GIPFwt induced significant stabilization ofβ-catenin in the cytosol from isolated crypt cells when compared to thatseen in the crypt cells from the control mice. FIG. 47A shows the levelof non-phosphorylated active β-catenin, and FIG. 47B shows the level oftotal p-catenin present in the cytosol. The non-phosphorylated β-cateninwas recognized by a β-catenin antibody that was purchased from Upstate(Waltham, Mass., USA), while the total β-catenin level was assayed usingan antibody from Abcam (Cambridge, Mass., USA), which recognizes boththe phosphorylated and non-phosphorylated protein. This result indicatesthat GIPF-induced crypt epithelial cell proliferation in mice may bemediated β-catenin signaling.

B. The effect of GIPFwt on the proliferation of isolated crypt cells wasassayed in vitro for the incorporation of ³H-thymidine. The resultsshowed that GIPFwt protein increased the proliferation of isolated cryptcells in a dose-dependent manner.

Therefore, the GIPF induces proliferation of isolated crypt cells bystabilizing β-catenin, and the isolated intestinal cells may be used toelucidate the signaling pathways that underlie the proliferative effectof GIPF.

C. The clonogenic assay described by Whitehead et al. (Whitehead et al.Gastroenterology, 117:858-865 (1999)) is performed to study the abilityof GIPF in controlling the proliferation and/or differentiation of theintestinal mucosa. In brief, an underlay containing an equal mixture of1% agar and 2×RPMI medium plus 10% FBS is added to 35 mm dishes.Isolated colonic crypt cells are added to top layer media (equal partsof 0.8% agarose and 2×RPMI medium plus 10% FBS) at 50,000 cells per mland 2 ml of cell suspension is aliquoted into each well. The plates areincubated in the presence of GIPF at 50, 100, and 200 ng/ml for 3-4weeks at 37° C. After the incubation, the plates are examined and thenumber of colonies are counted. Colonies are defined as aggregates ofmore than 40 cells.

GIPF stimulates colony formation. The clonogenic assay is used to testthe proliferative activity of GIPF and GIPF analogs in vitro.

Example 22 Effect of GIPF on TNBS-Induced Colitis

The hapten agent 2,4,6-trinitrobenzenesulfonic acid (TNBS) induces achronic colitis that is characterized by severe, transmural inflammationassociated with diarrhea, rectal prolapse, and weight loss. Theseclinical and histopathological features indicate that TNBS-inducedcolitis mimics important characteristics of human Crohn's disease(Neurath et al., J Exp Med 182:1281-1290 (1995)).

The therapeutic effect of GIPF was tested in mice with TNBS-inducedcolitis. Intestinal inflammation was induced in 6-8 week-old femaleBALBc mice (group 2) by a single rectal administration of 1 mg TNBS, asdescribed by Neurath et al, supra). A control animal group (Group 1)received rectal administration of vehicle alone (45% ethanol). Thetherapeutic effect of hGIPF was tested by administering subcutaneousdaily doses of 100 μg (group 3) or 50 μg (group 4) hGIPF (4 mg/kg or 2mg/kg) to animals that had received TNBS for 3 days. The mice weresacrificed after 7 days, and the induction of colitis by TNBS wasassessed. hGIPF significantly reduced the loss of body weight induced byTNBS in the animals of group 2 (FIG. 48). hGIPF also reduced the severediarrhea, ulceration, bleeding and atrophy of the colon that theTNBS-treated animals of groups 2 suffered (data not shown).

Histologic changes were evaluated in H&E stained paraffin-embeddedsections of the colon from the control and TNBS groups. hGIPF reducedthe TNBS-induced transmural infiltration and mucosal crypt structuraldisintegration in the mouse colon (FIG. 49). The graph in FIG. 49represents the effect of hGIPF determined by the histological grading ofcolonic colitis as follows:

Histological (microscopic) grading of colonic colitis SCORE CRITERIA 0Normal 1 Low level of (occasional) leukocyte infiltration, no structuralchanges 2 Moderate leukocyte infiltration in lamina pripria, surfaceepithelial lesion, no ulceration 3 High leukocyte infiltration withinflammatory cells extending into the submucosa, mucosal erosion, focalulceration, moderate thickening of the colon wall 4 Very high leukocyteinfiltration with transmural inflammation, extensive mucosal damage,loss of goblet cells, high vascular density, thickening of the colonwall, ulceration

In addition, hGIPF diminished the increase in TNBS-inducedmyeloperoxidase, which is a hallmark of neutrophil infiltration in themouse colon. GIPF treatment significantly reduces the TNBS-induceddiarrhea, inflammation and thickening of the colon wall, and loss ofgoblet cells, relative to the animals that are not treated with GIPF.Therefore, GIPF may potentially be used as a therapeutic to treatpatients with Crohns disease.

Example 23 Therapeutic Effect of GIPF on Chronic Dextran SulfateSodium-Induced Colitis in Mice

The efficacy of recombinant human GIPF (GIPFwt) in treating colitis wastested in a mouse model of chronic dextran sulfate sodium (DSS)-inducedcolitis (L'Heureux and Brubaker J Pharmacol Exp Ther 306:347-354 (2003);Kriegelstein et al., Clin Invest 110:1773-1782 (2002); Siegmund et al.,J Pharmacol Exp Ther 296:99-105 (2001)).

Six to eight-week old female BALB/c mice (Charles River Laboratories,Wilmington, Mass., USA) were housed in ventilated cages and acclimatedfor one week to a 12 hour light:dark cycle. Mice were fed 4% DSS (v/w)in drinking water from Day 0 to 7 to induce colitis. From Day 7 to Day21, mice were given water without DSS to induce the 1^(st) remissionphase. From Day 21 to Day 28, mice were again given 4% DSS to induce the1^(st) relapse phase. From Day 28 to Day 35, mice were again given waterwithout DSS to induce the 2^(nd) remission phase. On Day 35, mice wererandomized into various experimental groups and GIPF therapy was startedon Day 35 and continued to Day 42. Mice were monitored daily from Day 35to Day 42 for signs of disease activity. On Day 42, the experiment wasterminated, the mice were sacrificed and intestinal tissue was harvestedfor analysis.

GIPF significantly reduced DSS-induced colitis in mice in adose-dependent fashion as reflected by a significant decrease in theinflammatory bowel disease activity index (IBDAI) (FIG. 50; *P<0.05(ANOVA, DSS/Saline vs. DSS/hGIPF groups); #P<0.05 (ANOVA, DSS/Saline vs.DSS/KGF); **P<0.05 (ANOVA, DSS/Saline vs. DSS/GLP-2)). The definition ofIBDAI is given in Example 14. H&E stained sections of the smallintestine and colon showed that hGIPF prevented the DSS-induced damageto the intestinal mucosa of the mice, and reversed the DSS-inducedshortening of the villus height and crypt depth (FIG. 51). GIPF alsosuperseded the suppressive effect of DSS on the proliferation of cryptcells in the small intestine (FIG. 52; *P<0.05 (ANOVA, DSS/Saline vs.Water/Saline); #P<0.05 (ANOVA, DSS/hGIPF vs. DSS/Saline)

**P<0.05 (ANOVA, DSS/KGF vs. DSS/Saline); ##P<0.05 (ANOVA, DSS/GLP-2 vs.DSS/Saline)). The crypt proliferative index is defined in Example 14.

In summary, therapeutic treatment of GIPF significantly reduces chronicDSS-induced colitis in mice, indicating that GIPF may be a potentiallyuseful therapy to treat human inflammatory bowel disease.

Example 24 Reduction of 5-FU-Induced Toxicity by hGIPF

The efficacy of hGIPF in reducing the gastrointestinal toxicity of 5-FUwas evaluated in normal BDF-1 mice.

Mice were divided into the following groups:

1) Vehicle injected with saline treatment

2) 5-FU injected with saline treatment

3) 5-FU injected with GIPF treatment

Female BDF-1 mice, at age of 11-13-week, were given daily subcutaneousinjections of either saline or 100 μg per dose hGIPF beginning at Day-3.From Day 0 to Day 4, each mouse was injected intraperitoneally with adose of 50 mg/kg of 5FU for 4 consecutive days. Mice were monitored forbody weight, occurrence of diarrhea, and mortality on a daily basis.

GIPF treatment significantly reduced 5-FU-induced gastrointestinaltoxicity, including reducing maximum body weight loss, diarrhea score,and mortality (Table 9), thus indicating that GIPF is effective inreducing chemotherapy-induced gastrointestinal toxicity in mice.

TABLE 9 Toxicity Maximum TREAT- weight Diarrhea Mortality Survival 5-FUMENT loss (%) score (%) Time (day) YES NO 33.1 ± 3.6  2.8 ± 0.5  92 8.5± 1.2 YES hGIPF 12.5 ± 6.9* 0.9 ± 0.6* 8.3 10.0 ± 0.0* YES KGF 16.8 ±7.9* 1.7 ± 0.8* 25 9.0 ± 1.0 YES GLP-2 17.4 ± 8.3* 1.9 ± 0.8  42 8.4 ±1.1 *P < 0.05 (ANOVA, 5-FU/hGIPF, 5-FU/KGF or 5-FU/GLP-2 vs. 5_FU/saline

Example 25 In Vivo GIPF Activity in a Non-Human Primate

A repeat-dose study of the activity of GIPF was performed in Cynomolgusmonkeys to determine the activity of GIPF in a non-human primate.

Nine female non-naïve, monkeys were screened for health by SNBL USA(Everett, Wash., USA) staff veterinarian or a veterinary technician andunderwent hematology and serum chemistry screening. Of the nine femaleanimals confirmed healthy, 8 were selected and assigned to the studygroups. The animals, which were previously quarantined, were acclimatedto the study room at the SNBL USA facility for a minimum of 14 daysprior to initiation of the study.

Protocol: Eight females were assigned to four treatment groups asoutlined in the table below and dosed via intravenous bolus injection ofGIPF protein once daily for three days. On the fourth day, all animalswere administered one intravenous bolus injection of bromodeoxy uridine(BrdU) (50 mg/kg) approximately 4 hours prior to necropsy. Selecttissues were collected at necropsy.

Study Design Dose Dose Levels Concentration volume Animal Group (mg/kg)(mg/mL) (mL/kg) number 1   0 mg/kg 0 3.3 mL/kg 2 (control) 2 0.1 mg/kg1.5 mg/mL 0.067 mL/kg  2 3 1.0 mg/kg 1.5 mg/mL 0.67 mL/kg  2 4 5.0 mg/kg1.5 mg/mL 3.3 mL/kg 2Observations and Examination: Clinical observations were made duringacclimation and throughout the study as follows. Mortality and stoolchecks were performed once daily in the morning and clinicalobservations for general health and appearance were performed once dailyin the afternoon beginning at the start of acclimation to the end ofin-life. Additional clinical observations were performed, if necessaryand recorded. A staff veterinarian or veterinary technician evaluatedeach animal if clinical observations indicate a declining condition andthe Study Director were notified.

Blood sample collection: Blood was collected once during acclimation andonce on the day of necropsy before administration of BrdU for hematologyand serum chemistry.

Gross pathology examination: At necropsy, the external surfaces of thebody, all orifices, and the cranial, thoracic, and abdominal cavitiesand their contents were examined. Organ Weight and histopathologyexamination were performed macroscopically, collected and preserved in10% neutral buffered formalin for histopathologic examination. Examinedtissues are listed below.

Brain Large Intestine^(a) Small Intestine^(a) brain stem cecum jejunumcerebellum colon duodenum cerebrum rectum ileum Spleen Liver Tongue

Tissue proliferation assay by using BrdU IHC: paraffin-embedded sectionswere prepared for BrdU assay.

Result: Gross pathological examination at necropsy found no obviouschanges in examined organs suggesting no acute toxicity of hGIPF in thistreatment regimen. In addition, hematology and serum chemistry on bloodsamples collected before and after hGIPF treatment demonstrated nochanges in blood cell components as well as tested serum biochemistryparameters.

There appears to be a dose-related increase in the length of the smallintestine. Average intestinal length (in cm) for groups 1, 2, 3, and 4was 120.65, 122.555, 133.350, and 142.875 respectively. In addition, assummarized in the table below (Table 10), microscopic evaluation ofhistology of individual tissues demonstrated crypt hyperplasia ofduodenum, jejunum and ileum in all GIPF treated groups. Hyperplasia ofcrypts was also observed in cecum, colon and rectum in a dose relatedmanner. This result suggests that hGIPF has a proliferative effect oncrypt epithelial cells of monkey intestine that is consistent with thatobserved in mouse and rat.

To confirm the proliferative effect of hGIPF, immunohistochemistry wasperformed to analyze the incorporation of BrdU in the small and largeintestine. hGIPF increases BrdU positive proliferation index in bothsmall intestine and colon.

These finding show that hGIPF increases the proliferation of the cryptepithelium in a non-human primate.

Individual Histopathology Findings

Grade

_: No abnormal changes

±: Very slight

+: slight

2+: Moderate

3+: Marked

TABLE 10 Individual Histopathology Findings in Female Cynomolgus MonkeysGroups (animal number) 1 2 3 4 Tissue Findings #1 #2 #1 #2 #1 #2 #1 #2Duodenum Crypt − − − ± ± ± + 2+ hyperplasia Jejunum Crypt − − ± ± ± ±± + hyperplasia Ileum Crypt − − − ± + ± + + hyperplasia Cecum Crypt − −− − ± ± + + hyperplasia, increased − − − − − − ± + gland length ColonCrypt − − − − ± ± ± + hyperplasia, increased − − − − − − − ± glandlength Rectum Crypt − − − − ± + ± + hyperplasia Liver — − − − − − − − −Spleen — − − − − − − − − Cerebrum — − − − − − − − − Cerebellum — − − − −− − − − Brain stem — − − − − − − − −

Example 26 Adsorption Distribution Metabolism Excretion (ADME) StudyUsing Radioactively Labeled ¹²⁵I-hGIPF Protein

Study aim: To determine the plasma pharmacokinetics and tissuedistribution of [¹²⁵I]-hGIPF in mice.Protein labeling: hGIPF protein was labeled with [¹²⁵I] by IODO-GENlabeling method (Amersham). The initial specific activity upon labelingwas 35 uCi/ug (1020 Ci/mmol). Labeled protein was further purified priorto injection into mice.Animals: male CD-1® [Crl:CD-1® (ICR) BR mice were acclimated for 7 daysprior to the injection and housed individually in clean suspendedwire-mesh cages. The cages were elevated above cage-board or othersuitable material, changed at least three times each week. Each mousewas given a 1.67 mg/kg dose of hGIPF that contained 3 μCi of ¹²⁵I-hGIPFprotein. After receiving the radiolabeled dose, animals that werescheduled for collection of urine and feces were housed individually inmetabolism units.

Study Design:

Number of Total Animals Number Samples Dosage Dosage Times of per Timeof Group Collected Level Volume Euthanasia Point Animals 1 Blood and1.67 mg/kg 10 mL/kg 5, and 30 min 3 18 Tissues and 1, 3, 6, and 24 hrpost-dosing 2 Urine, Feces, 1.67 mg/kg 10 mL/kg 24 hr post- 3 3 Tissuesand dosing Carcass

All animals received for this study were treated with sodium iodide toblock uptake by the thyroid of free iodide derived from the labeled testarticle. An oral (gavage) administration of 0.1 mL of 1% NaI solutionwas given at approximately 48, 24 and 1 hours before dosing with theradiolabeled protein. Each animal received a single dose of [¹²⁵I]-hGIPFthat was administered via an intravenous injection. The animals weredivided into two groups and analyzed as outlined in the Study Designabove. At the indicated times for euthanasia, blood samples werecollected, and the cellular fraction and plasma were separated foranalysis. Tissue samples of liver, kidney, lung, tongue, spleen, brain,esophagus, stomach, small intestine, large intestine, and largeintestine including its contents were collected, and the incorporationof [¹²⁵I]-hGIPF in the tissues was determined.

Analyses for the incorporation of ¹²⁵I-hGIPF was performed by gammacounting in a DPC GAMMA-C12 multicrystal gamma counter according to WILStandard Operating Procedures. The results of gamma counting werecorrected for isotopic half-life. Calculations of the amounts ofradioactivity in various materials generated in the study were performedusing programs of the WIL Toxicology Data Management System orspreadsheets according to WIL Standard Operating Procedures. Generally,only descriptive statistics (e.g., totals, arithmetic means, standarddeviations, standard errors, coefficients of variation, percentages)were used. Where possible, standard pharmacokinetic parameters (e.g.,C_(max), t_(max), AUC (Area Under the Curve), T_(1/2) (half-life)) werecalculated using standard pharmacokinetic equations.

Results: The data showing the concentration and the kinetics of[¹²⁵I]-hGIPF in mouse plasma, red blood cells, liver, kidney, lung,heart, brain, spleen, esophagus, stomach, small intestine, and largeintestine are shown in Tables 11-14.

TABLE 11 CONCENTRATION AND KINETICS OF [¹²⁵I]-hGIPF EQUIVALENTS IN MOUSEPLASMA AND RED BLOOD CELLS FOLLOWING INTRAVENOUS ADMINISTRATION AT 1.67MG/KG Plasma Red Blood Cells Time (ng/g) (ng/g) (hr) (Mean ± SD) (Mean ±SD)  0.083 11145 (113)  2745  0.5 3894 (485) 2121  1 2506 (490) 1658  31347 (68)  971  6  476 (204) 295 24 60 (7) 23 C_(max) (ng/g) 11145 2745t_(max) (h) 0.083 0.083 AUC₀₋₂₄ (ng-h/g) 16607 9458 Terminal PhaseKinetics (Linear Regression of Log Concentration vs. Time from 3-24 hr)Slope (b) −0.05939 −0.07225 Y-Intercept (ng) 1517.75 1161.79 Coefficientof Determination (r²) 0.960 0.967 Elimination Rate Constant (h⁻¹) 0.13670.1664 Half-life (h) 5 4 N = 3 except 24 h, N = 6.

TABLE 12 CONCENTRATION AND KINETICS OF [¹²⁵I]-hGIPF EQUIVALENTS IN MOUSELIVER, KIDNEY, LUNG, HEART, BRAIN, AND SPLEEN FOLLOWING INTRAVENOUSADMINISTRATION AT 1.67 MG/KG Liver Kidney Lungs Tongue Brain Spleen(ng/g) (ng/g) (ng/g) (ng/g) (ng/g) (ng/g) (Mean ± SD) (Mean ± SD) (Mean± SD) (Mean ± SD) (Mean ± SD) (Mean ± SD) 0.083 hr 9104 (959) 24581(5032) 3332 (462) 1157 (88)  141 (25) 1956 (354)  0.5 hr  4982 (1319)21283 (4731) 1997 (375) 1124 (202) 105 (28) 1905 (663)    1 hr 3635(413) 17039 (1543) 1575 (375) 1044 (292)  95 (18) 1380 (106)    3 hr2706 (185) 13445 (1084)  633 (450) 588 (32) 51 (7) 840 (74)    6 hr 1757(213) 8933 (800)  337 (106) 227 (91) 17 (9)  457 (193)   24 hr 875 (75)4707 (485)  63 (13) 35 (7)  5 (3) 157 (49) C_(max) (ng/g) 9104 245813332 1157 141 1955.54 t_(max) (h) 0.083 0.083 0.083 0.083 0.083 0.083AUC₀₋₂₄ (ng- 42191 206968 9404 6282 554 11400 h/g) Terminal PhaseKinetics (Linear Regression of log Concentration vs. Time from 3-24 hr)Slope (b) −0.02106 −0.01953 −0.04512 −0.05344 −0.04209 −0.03164Y-Intercept 2742.03 13559.24 746.08 649.87 47.18 873.39 (ng) Coefficientof 0.936 0.932 0.982 0.958 0.883 0.947 Determination (r²) Elimination0.0485 0.0450 0.1039 0.1230 0.0969 0.0728 Rate Constant (h⁻¹) Half-life(h) 14 15 7 6 7 10 N = 3 except 24 h, N = 6.

TABLES 13 A AND B CONCENTRATION AND KINETICS OF [¹²⁵I]-hGIPF EQUIVALENTSIN MOUSE ESOPHAGUS, STOMACH, SMALL INTESTINE, AND LARGE INTESTINEFOLLOWING INTRAVENOUS ADMINISTRATION AT 1.67 MG/KG Small Intestine LargeIntestine Esophagus (ng/g) Stomach (ng/g) (ng/g) (ng/g) (Mean ± SD)(Mean ± SD) (Mean ± SD) (Mean ± SD) A 0.083 hr 1560 (321) 1960 (166) 1117 (135) 1016 (150)  0.5 hr 1743 (178) 2855 (1140) 1191 (423)  910(135)    1 hr 1666 (743) 5545 (3546) 1053 (369) 1006 (189)    3 hr 1199(330) 3678 (2047) 664 (28)  694 (141)    6 hr 350 (63) 1021 (475)   222(110)  383 (175)   24 hr  48 (43) 106 (40)  32 (6)  74 (13) C_(max)(ng/g) 1743 5545 1191 1016 t_(max) (h) 0.5 1 0.5 0.083 AUC₀₋₂₄ (ng-h/g)10372 29604 6421 8344 B Terminal Phase Kinetics (Linear Regression oflog Concentration vs. Time from 3-24 hr) Slope (b) −0.06022 −0.06683−0.05745 −0.04408 Y-Intercept (ng) 1247.25 3997.67 715.76 822.73Coefficient of 0.936 0.947 0.948 0.984 Determination (r²) EliminationRate 0.1387 0.1539 0.1323 0.1015 Constant (h⁻¹) Half-life (h) 5 5 5 7**N = 3 except 24 h, N = 6.

TABLES 14 A AND B RECOVERY OF hGIPF EQUIVALENTS FROM MICE 24 HOURS AFTERIV ADMINISTRATION AT 1.67 MG/KG A % GIPF/Intestinal Total Anim. No. %GIPF/Tissues Contents % GIPF/Urine % GIPF/Feces % GIPF/Carcass Recovery†1 7.43 0.13 85.70 3.48 1.87 98.6 2 7.61 0.13 76.70 3.12 1.82 89.4 3 7.030.32 87.47 3.53 1.76 100.1 Mean: 7.35 0.19 83.29 3.38 1.82 96.0 SD: 0.300.11  5.77 0.23 0.06 5.81 B As expected, the earliest T_(max) and thegreatest C_(max) were observed in highly perfused tissues i.e. liver,kidney, spleen and lung, and the longest half life was seen in kidneyand liver (Table 11). Tissues Liver Small Int. Colon Brain Spleen LungKidney Stomach Esoph. Tongue Sum 2.39 0.07 0.06 0.01 0.02 0.02 4.83 0.030.01 0.01 7.43 2.13 0.10 0.06 0.00 0.02 0.02 5.17 0.10 0.00 0.01 7.612.64 0.07 0.04 0.01 0.02 0.02 4.21 0.03 0.00 0.01 7.03 †Sum of tissues,GI contents, urine including cage rinse, feces, and carcass

The tissues of the gastrointestinal tract including esophagus, stomachand small intestine each displayed a protracted T_(max) (Table 12).Table 13 A shows the percent recovery of radiolabeled GIPF in tissuesfrom various organs and in the intestinal contents, urine, feces andcarcass of animals 24 hours after administration of radiolabeled hGIPF.The recovery of hGIPF from individual organ tissues is given in Table13B. The data show that the distribution of radiolabeled hGIPF isunusually high for organs of the gastrointestinal tract followingadministration via the intravenous route, thus suggesting that hGIPF mayhave a high affinity for gastrointestinal tissues.

Example 27 Irradiation-Induced Mucositis Evaluation of Optimal TreatmentRegimen

The objective of this study was to define a therapeutic protocol thatwould provide the maximum prophylactic effect of GIPF againstirradiation-induced mucositis.

Adult male BDF-1 mice 10˜12 weeks of age at the time of use. The animalswere housed for 1 week on a 12 hr light/dark cycle and were allowed foodand water ad libitum throughout. Animals were randomly divided into 6groups of 5 animals each (total 30 mice) and were treated as follows:

-   -   1. Untreated, unirradiated control.    -   2. 4 mg/kg hGIPF iv at 3, 2, 1 day prior to 13 Gy X-ray exposure        (whole body irradiation).    -   3. 4 mg/kg hGIPF iv at 4, 3, 2 days prior to 13 Gy X-ray        exposure (whole body irradiation).    -   4. 4 mg/kg hGIPF iv 5, 4, 3 days prior to 13 Gy X-ray exposure        (whole body irradiation).    -   5. 4 mg/kg hGIPF iv 6, 5, 4 days prior to 13 Gy X-ray exposure        (whole body irradiation).    -   6. saline iv 72, 48, 24 hours prior to 13 Gy X-ray exposure        (whole body irradiation).

Animals were exposed to whole body irradiation with single dose of 13 GyX-ray delivered at 2.7 Gy/min. Animals were inspected daily afterirradiation.

4 days after irradiation the animals were sacrificed. Two hours prior tosacrifice, animals were injected with 4 mg BrdU in a volume of 0.4 ml.Upon dissection, the length and weight of small and large intestine weremeasured and ˜1 cm of tissue segments of the small intestine(mid-jejunum), colon (transverse colon), tongue, esophagus and stomachwere fixed in 10% formaline.

Cross sections of the small intestine were analyzed for BrdU uptake. Thenumber of surviving crypts per section were scored and the average pergroup determined. Only crypts containing 10 or more strongly H&E stainedcells (excluding Paneth cells) and only intact sections devoid of Peyerspatches were scored.

The average crypt width (measured at its widest point) was also measuredin order to correct for scoring errors due to crypt size difference. Thecorrection is applied thus:

$\frac{{Corrected}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {crypts}}{{cross}\mspace{14mu} {section}} = {\frac{{Mean}\mspace{14mu} {crypt}\mspace{14mu} {width}\mspace{14mu} {in}\mspace{14mu} {untreated}\mspace{14mu} {control}}{{Mean}\mspace{14mu} {crypt}\mspace{14mu} {width}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {animal}} \times {Mean}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {surviving}\mspace{14mu} {crypts}\mspace{14mu} {in}\mspace{14mu} {treatment}\mspace{14mu} {group}}$

Results: The effect of hGIPF on the survival of crypts followingexposure to radiation is shown in FIG. 53. The data show that hGIPFsignificantly reduced radiation-induced intestinal mucositis when hGIPFwas administered to the animals at 24 hr or 48 hr prior to the totalbody irradiation (Table 15).

These findings confirm that hGIPF may be used as a prophylactic tooffset the deleterious effects of radiation-induced intestinalmucositis, and that dosing at 24 hours prior to total body irradiationprovides the greatest protection to the intestinal crypts.

TABLE 15 hGIPF therapy Irradiation dose Treatment hGIPF protection (Gy)GIPF (mg/kg/day) schedule factor (mean ± SD) 13 None None 1.0 ± 0.5 13 4Day −3 to −1 15.8 ± 6.2* 13 4 Day −4 to −2  9.4 ± 6.8* 13 4 Day −5 to −34.8 ± 3.3 13 4 Day −6 to −4 5.3 ± 4.4 *P < 0.05 (ANOVA, 13 Gy/hGIPF vs.13 Gy/Saline)

Example 28 Cell Lineage-Dependent Proliferation Assay in Mouse SmallIntestine

The effect of hGIPF on intestinal crypts was studied to determinewhether the effect of hGIPF prior to the onset of morphological changesoccurs by affecting either of both the stem cells and the transitionalproliferating cells of the crypt.

Animals were randomly divided into the following groups:

-   -   1. PBS (1 mouse per group): 1, 3, 6, 12, 24 or 48 hr after        injection    -   2. hGIPF (2 mice per group, 100 ug single injection): 1, 3, 6,        12, 24 or 48 hr after injection        Each animal was injected with BrdU (4 mg/kg) 2 hour prior to        sacrifice.        Crypt depth, crypt proliferation index, and cell positional        proliferation analysis were performed in mid jejunal sections of        small intestine. The crypt proliferative index was measured by        BrdU immunohistochemistry at the indicated times (3, 6, 12, 24        and 48 hours) after single injection of hGIPF (100 ug). 40        crypts from 2 mice were analyzed for BrdU incorporation and the        results are given as the mean±SD (*P<0.01, ANOVA).        Results: As shown in Table 16, hGIPF increased proliferation of        small intestinal crypt cells as early as 3 hours and the        proliferation reached a peak at 24 hours following hGIPF        treatment. hGIPF-induced crypt proliferation was reversed within        48 hours. In addition, positional analysis of the BrdU positive        cells (Potten et al., Int J Exp Path 78:219-243 (1997))        demonstrated a significant increase in the proliferation of        crypt cells at position 3˜5 (from the bottom of crypts where        stem cells are located) as well as upper part of the crypts.

These data suggest that hGIPF may affect both stem cells and thedividing transit cell population.

TABLE 16 % BrdU positive cells % BrdU positive cells TIME (hr) PBScontrol group hGIPF group 3  38.0 ± 12.47  47.7 ± 8.38 6 36.45 ± 8.3349.75 ± 11.3 12 39.16 ± 8.57 51.24 ± 9.86 24 36.55 ± 9.62 74.97 ± 9.0 48  33.0 ± 5.32 19.5 ± 6.5

Example 29 Effect of hGIPF on the Differentiation and Migration CryptCells

The number of Goblet and Paneth cells was scored following treatment ofmice with hGIPF to determine whether hGIPF affects the population anddistribution of these cell types in the small intestine.

Alcian blue staining was performed to visualize Goblet cells in midjejunal sections from PBS and hGIPF-treated mice (n=3). Animals weregiven daily injections of hGIPF (100 ug) or PBS for 3 or 7 days. Tovisualize Paneth cells, immunohistochemistry (IHC) was performed on themid jejunal sections of the same animals using anti-lysozyme antibody.

Results: Immunohistochemical analysis and Alcian blue staining of smallintestine demonstrated no significant changes in Paneth cells and Gobletcells numbers in the small intestine of hGIPF treated mice. hGIPF didnot affect the maturation and migration of differentiated cells alongthe crypt/villus axis.

Example 30 Transgenic Chimaeric Mice that Express hGIPF in IntestinalEpithelial Cells 1. Preparation of Long Fragment of Mouse Villin GenePromoter (FIG. 54A)

Villin, an actin bundling protein found in the apical brush border ofabsorptive tissues, is one of the first structural genes to betranscriptionally activated in the embryonic intestinal endoderm. In theadult, villin is broadly expressed in every cell of the intestinalepithelium on both the vertical axis (crypt to villus tip) and thehorizontal axis (duodenum through colon) of the intestine. Madison etal. documented that a 12.4 kb region of the mouse villin gene driveshigh level expression of two different reporter genes (LacZ and Crerecombinase) within the entire intestinal epithelium of transgenic mice(J. Biol. Chem. 277, p 33275-33283, 2002). To generate transgenicchimaeric mice expressing human GIPF in intestinal epithelial cells weconstructed a expression vector in which the GIPF cDNA is linked to thistranscriptional regulatory sequences directing its expression inintestinal epithelial cells.

Nucleotide sequence information of upstream region of mouse villin genewas obtained from public database (ensembl). Mouse BAC (RP23-278N11;GenBank Accession Number: AC098570) DNA was digested with EcoRI andBamHI (Roche) and subjected to 0.8% agarose gel electrophoresis toisolate an approximately 11 kb fragment. Following the digestion ofpBluescriptIISK(−) (STRATAGENE) with EcoRI and BamHI (Roche), the vectorfragment was isolated by 0.8% agarose gel electrophoresis and treatedwith calf intestine alkaline phosphatase to dephosphorylate its bothends. The above approximately 11 kb DNA fragment was ligated to thedephosphorylated vector fragment and the ligation mixture wastransfected to XL10-Gold Ultracompetenet Cells (STRATAGENE). DNA samplesprepared from the resultant transformants was subjected to PCRamplification using the primer set described below (SEQ ID NO: 120 and121). Sequence analysis of the amplified fragment showed the inclusionof an approximately 11.2 kb of mouse villin gene promoter fragment(pPvil 11.2). The pPvil 11.2 was digested with the restriction enzymes,ClaI and BamHI, and the reaction mixture was subjected to 0.8% agarosegel electrophoresis to isolate approximately 11.2 kb fragment.

PvilEIBI-FW1 (SEQ ID NO: 120) GATCAGCAGCTGGAACAAACACAG PviLEIBI-RV1 (SEQID NO: 121) TGCACAATCAGTCAATCAACAGAGC

(2) Preparation of Short Fragment of Mouse Villin Gene Promoter (FIG.54B)

Based on the nucleotide sequence of mouse villin gene upstream regionobtained from the public database (ensembl), two synthetic DNAs weresynthesized (SEQ ID NO: 122 and 123).

PvilBI-FW (SEQ ID NO: 122) GGCGGATCCCTGAGTTGGAGGCCAGTTTGGPvilBI-NcoIXbalRV (SEQ ID NO: 123)GCTCTAGACCATGGTGGACGAGCCTAGAGGAGAAGGCAT

KOD-puls (TOYOBO) was used for the PCR reaction. The PCR reactionmixture contained 10 pmole of each primer and mouse BAC (RP23-278N11;GenBank Accession Number: AC098570) DNA as a template. This PCRamplification was performed using an initial denaturing incubation at94° C. for two minutes. Then 30 cycles of denaturation, annealing andamplification were performed by incubation at 94° C. for 15 sec and 68°C. for two minutes. A PCR product (approximately 1.9 kb) was purified by0.8% agarose gel electrophoresis and QIAquick Gel Extraction Kit(QIAGEN). Following the digestion of an isolated PCR product with BamHIand XbaI, the digested fragment was purified by 0.8% agarose gelelectrophoresis and QIA quick Gel Extraction Kit (QIAGEN). The purifiedfragment was ligated to pBluescriptIISK(−) (STRATAGENE) that wasdigested with XhoI and XbaI, and treated with calf intestine alkalinephosphatase to dephosphorylate its both ends. The ligation mixture wastransfected to DH5α and the DNA samples prepared from the resultanttransformants were analyzed by nucleotide sequencing to confirm thestructure of inserted fragment. The clone including a fragment with acorrect nucleotide sequence was digested with NcoI. Following thetreatment of digested fragment with Klenow fragment (TAKARA BIO) forblunting its both ends, it was further digested with XbaI and purifiedby 0.8% agarose gel electrophoresis. The resultant fragment was treatedwith E. Coli C₇₋₅ alkaline phosphatase to dephosphorylate its both ends.

(3) Preparation of GIPF Fragment (FIG. 54C)

Hy01XhlSphlFW (SEQ ID NO: 124) CCGCTCGAGGCATGCGGCTTGGGCTGTGTGTGGTGGCCCTGHy01BgXb-RV (SEQ ID NO: 125)GCTCTAGAAGATCTCTAGGCAGGCCCTGCAGATGTGAGTGGCCC

KOD-puls-(TOYOBO) was used for the PCR reaction. The PCR reactionmixture contained 10 pmole of each primer (SEQ ID NO: 124 and 125) andthe GIPF cDNA as a template. This PCR amplification was performed usingan initial denaturing incubation at 94° C. for three minutes. Then 30cycles of denaturation, annealing and amplification were performed byincubation at 94° C. for 15 sec and 68° C. for two minutes. A PCRproduct (approximately 800 bp) was purified by electrophoresis using0.8% agar and QIAquick Gel Extraction Kit (QIAGEN). Following thedigestion of isolated PCR product with XhoI and XbaI, it was ligated topBluescriptIISK(−) that was digested with XhoI and XbaI, and treatedwith calf intestine alkaline phosphatase to dephosphorylate its bothends. The ligation mixture was transfected to DH5α and the DNA samplesprepared from the resultant transformants were analyzed by nucleotidesequencing to confirm the structure of inserted fragment. The cloneincluding a fragment with a correct nucleotide sequence was digestedwith SphI. Following the treatment of digested fragment with Bluntinghigh (TOYOBO) for blunting its both ends, it was further digested withXbaI and purified by 0.8% agarose gel electrophoresis.

(4) Construction of pPvil 2-01 (FIG. 54D)

The GIPF fragment prepared in (3) was ligated to pPvil2 prepared in (2),and the ligation mixture was transfected to DH5α. The DNA samplesprepared from the resultant transformants were analyzed by nucleotidesequencing to confirm the structure of inserted fragment. The cloneincluding a fragment with a correct nucleotide sequence was selected(pPvil 2-01).

(5) Preparation of pIRES-GFP (FIG. 54E)

Following the digestion of pIRES2-EGFP (BD Bioscience Clontech) withEcoRI and NotI, the fragment including the IRES-GFP region was purifiedby 0.8% agarose gel electrophoresis and QIA quick Gel Extraction Kit(QIAGEN). The purified fragment (IRES-GFP) was ligated to pcDNA3(Invitrogen) that was digested with XhoI and XbaI, and treated with calfintestine alkaline phosphatase to dephosphorylate its both ends. Theligation mixture was transfected to DH5α and the DNA samples preparedfrom the resultant transformants were analyzed by nucleotide sequencingto confirm the structure of inserted fragment. The clone including afragment with a correct nucleotide sequence was selected (pIRES-GFP).

(6) Construction of pUC119 IRES-GFP (FIG. 54F)

Following the digestion of pIRES-GFP with BamHI and XbaI, the fragmentincluding the IRES-GFP region was purified by 0.8% agarose gelelectrophoresis and QIA quick Gel Extraction Kit (QIAGEN). The purifiedfragment (IRES-GFP) was ligated to pUC119 that was digested with BamHIand XbaI, and treated with calf intestine alkaline phosphatase todephosphorylate its both ends. The ligation mixture was transfected toDH5α and the DNA samples prepared from the resultant transformants wereanalyzed by nucleotide sequencing to confirm the structure of insertedfragment. The clone including a fragment with a correct nucleotidesequence was selected (pUC119 IRES-GFP).

(7) Construction of pUC119 IRES-GFP+As (FIG. 54G)

The DNA fragment prepared by annealing of synthesized oligonucleotidesdescribed below (SEQ ID NO: 126 and 127) was ligated to pUC119 IRES-GFPthat was digested with EcoRI and BamHI, and treated with calf intestinealkaline phosphatase to dephosphorylate its both ends. The ligationmixture was transfected to DH5α and the DNA samples prepared from theresultant transformants were analyzed by nucleotide sequencing toconfirm the structure of inserted fragment. The clone including afragment with a correct nucleotide sequence was selected (pUC119IRES-GFP+As).

El-BIAscl-(BI) S AATTCGGATCCGGCGCGCC (SEQ ID NO: 126) El-BIAscl-(BI) ASGATCGGCGCGCCGGATCCG (SEQ ID NO: 127)(8) Construction of pUC119 IRES-GFP+loxP (FIG. 54H)

The DNA fragment prepared by annealing of synthesized oligonucleotidesdescribed below (SEQ ID NO: 128 and 129) was ligated to pUC119IRES-GFP+As that was digested with NotI and XhoI, and treated with calfintestine alkaline phosphatase to dephosphorylate its both ends. Theligation mixture was transfected to DH5α and the DNA samples preparedfrom the resultant transformants were analyzed by nucleotide sequencingto confirm the structure of inserted fragment. The clone including afragment with a correct nucleotide sequence was selected (pUC119IRES-GFP+loxP).

Nt-PmloxP-Xh S (SEQ ID NO: 128)GGCCGTTTAAACATAACTTCGTATAATGTATGCTATACGAAGTTATC Nt-PmloxP-Xh AS (SEQ IDNO: 129) TCGAGATAACTTCGTATAGCATACATTATACGAAGTTATGTTTAAAC

(9) Preparation of Bovine Growth Hormone (BGH) PolyA Fragment (FIG. 54I)

BGHpAFW (SEQ ID NO: 130) CGGGATCCGTTTAAACCTGTGCCTTCTAGTTGCCAGCGATCBGHpARV (SEQ ID NO: 131) CGGATATCCCATAGAGCCCACCGCATCCCCAGC

KOD-puls-(TOYOBO) was used for the PCR reaction. The PCR reactionmixture contained. 10 pmole of each primer (SEQ ID NO: 130 and 131) andthe IRES-GFP fragment prepared in (6) as a template. This PCRamplification was performed using an initial denaturing incubation at94° C. for three minutes. Then 30 cycles of denaturation, annealing andamplification were performed by incubation at 94° C. for 15 sec and 68°C. for two minutes. A PCR product (approximately 0.2 kb) was purified by0.8% agarose gel electrophoresis and QIAquick Gel Extraction Kit(QIAGEN). Following the digestion of isolated PCR product with BamHI andEcoRV, it was ligated to pBluescriptIISK(−) that was digested with BamHIand EcoRV, and treated with calf intestine alkaline phosphatase todephosphorylate its both ends. The ligation mixture was transfected toDH5α and the DNA samples prepared from the resultant transformants wereanalyzed by nucleotide sequencing to confirm the structure of insertedfragment. The clone including a fragment with a correct nucleotidesequence was digested with PmeI and EcoRV, and the fragment includingthe bovine growth hormone (BGH) polyA region was purified byelectrophoresis using 0.8% agar and QIAquick Gel Extraction Kit(QIAGEN).

(10) Preparation of DNA Fragment Including IRES-GFP, Bovine GrowthHormone PolyA and loxP Sequences (FIG. 54J)

The pUC119 IRES-GFP+loxP was digested with PmeI and purified by 0.8%agarose gel electrophoresis. The BGH polyA fragment prepared in (9) wasligated to the purified pUC119 IRES-GFP+loxP vector that was treatedwith calf intestine alkaline phosphatase to dephosphorylate its bothends. The ligation mixture was transfected to DH5α and the DNA samplesprepared from the resultant transformants were analyzed by nucleotidesequencing to confirm the structure of inserted fragment. The cloneincluding a BGH polyA fragment in a same direction to coding sequence ofGFP was selected (pIRES-GFP+pA). The pIRES-GFP+pA was digested withBamHI and XbaI, and the fragment including the IRES-GFP, bovine growthhormone polyA and loxP sequences was purified by electrophoresis using0.8% agar and QIAquick Gel Extraction Kit (QIAGEN).

(11) Construction of pPvil 2-01GFP (FIG. 54K)

The DNA fragment including the IRES-GFP, bovine growth hormone polyA andloxP sequences [see (10)] was ligated to pPvil2GIPF that was digestedwith BgIII and XbaI, and treated with calf intestine alkalinephosphatase to dephosphorylate its both ends. The ligation mixture wastransfected to DH5α and the DNA samples prepared from the resultanttransformants were analyzed by nucleotide sequencing to confirm thestructure of inserted fragment. The clone including a fragment with acorrect nucleotide sequence was selected (pPvil 2-01 GFP).

(12) Construction of pPv-Total (FIG. 54L)

The approximately 11.2 kb of long fragment of mouse Villin gene promoter[see (1)] was ligated to pPvil2-01 GFP that was digested with BgIII andClaI, and treated with E. coli C75 alkaline phosphatase todephosphorylate its both ends. The ligation mixture was transfected toXL10-Gold Ultracompetent Cells (STRATAGENE) and the DNA samples preparedfrom the resultant transformants were analyzed by nucleotide sequencingto confirm the structure of inserted fragment. The clone including thefragment with a correct nucleotide sequence was selected (pPv-total).

(13) Construction of pLoxP-STneoR (FIG. 54M)

The pLoxP-STneo described in WO 00/10383 was digested with XhoI andtreated with Blunting high (TOYOBO) for blunting its both ends. Theresultant DNA fragment including loxP-Neo^(r)-loxP unit was purified by0.8% agarose gel electrophoresis. The DNA fragment prepared by annealingof synthesized oligonucleotides described below (SEQ ID NO: 132 and 133)was ligated to pBlueLAB (WO 00/10383) that was digested with PacI andFseI, and purified by 0.8% agarose gel electrophoresis. The ligationmixture was transfected to DH5α and the DNA samples prepared from theresultant transformants were analyzed by nucleotide sequencing toconfirm the structure of inserted fragment. The clone including afragment with a correct nucleotide sequence was selected (pBlueLAB2).The above DNA fragment including loxP-Neo^(r)-loxP unit was ligated tothe pBlueLAB2 vector that was digested with EcoRV, and was treated withcalf intestine alkaline phosphatase to dephosphorylate its both ends.The ligation mixture was transfected to DH5α and the DNA samplesprepared from the resultant transformants were analyzed by nucleotidesequencing to confirm the structure of inserted fragment. The cloneincluding the fragment in an opposite direction to pLoxP-STneo (WO00/10383) was selected (pLoxP-STneoR).

AsiSl-S (SEQ ID NO: 132) □TAACCGCGATCGCGGCCGG AsiSl-AS (SEQ ID NO: 133)□CCGCGATCGCCCTTAAT(14) Construction of pPv01GFP (FIG. 54N)

The pPv-total plasmid DNA was digested with restriction enzymes, ClaIand XhoI, and the DNA fragment including Pv-GIPF unit was purified by0.8% agarose gel electrophoresis. The purified DNA fragment was ligatedto pLoxP-StneoR that was digested with ClaI and XhoI, and treated withcalf intestine alkaline phosphatase to dephosphorylate its both ends.The ligation mixture was transfected to XL10-Gold Ultracompetent Cells(STRATAGENE) and the DNA samples prepared from the resultanttransformants were analyzed by nucleotide sequencing to confirm thestructure of inserted fragment. The clone including a fragment with acorrect nucleotide sequence was selected (pPv01 GFP).

(15) Preparation of pPv01GFP Plasmid DNA for Electroporation to Mouse ESCells

The plasmid DNA of pPv01GFP (60 μg) was digested with ClaI in thereaction mixture containing 1 mM spermidine (pH7.0, Sigma) for 5 hoursat 37° C. The reaction mixture was then subjected to phenol/chloroformextraction and ethanol precipitation (0.3M NaHCO₃) for 16 hours at −20°C. The linearized vector fragment was dissolved in HBS buffer (0.5μg/μl) and used for the following electroporation experiments.

(16) Production of Transgenic Chimaeric Mice Expressing Human GIPF andGFP in Intestinal Epithelial Cells

General procedures for obtaining mouse embryos, cultivation, injectionof the ES cells into the embryos, transplantation to the uteri of fostermothers were carried out in accordance with the method described inShinichi Aizawa, “Biomanual Series 8, Gene Targeting”, published byYodosha, 1995.

The linearized pPv01GFP vector was transfect into C57BL/6×CBA F1 strainderived mouse TT2F ES cells ((Uchida, 1995), Lifetech oriental) byelectroporation according to the method described by Shinichi Aizawa,“Biomanual Series 8, Gene Targeting”, published by Yodosha, 1995. Theelectroporated ES cells were suspended in 20 ml of ES medium andinoculated into two 100 mm tissue culture plastic plates (Corning) intowhich feeder cells were seeded in advance. After one day, the medium wasreplaced with a medium containing a 200 μg/ml of G418 (Invitrogen).Seven to nine days thereafter, a total of 24 colonies for each vectorwere picked up. Each colonies was grown up to confluence in a 12-wellplate, and then four fifth of the culture was suspended in 0.2 ml ofcryopreservation medium (ES medium+10% DMSO (Sigma)) and stored frozenat −80° C. The remaining one fifth was inoculated into a 12-well gelatincoated plate and cultured for 2 days. Then, genomic DNA was isolatedusing the Puregene DNA Isolation Kit (Gentra System). Genomic DNAisolated from G418 resistant TT2F cells was digested with restrictionenzymes EcoRI and XhoI and then subjected to 0.8% agarose gelelectrophoresis. Using EcoRI-XhoI digestion, retention of an intactexpression unit including Villin promoter, human GIPF cDNA, GFP cDNA andBGH polyA sequences of pPv01GFP in the G418-resistant clones can bedetermined by the detection of an approximately 16 kb band. SeparatedDNA fragments were transferred to a membrane (Gene Screen, NEN Lifescience Products) and then hybridization was carried out using the DNAfragment as probe prepared from IRES region of pPV01GFP [see (13)] byPCR using a primer set as described below (SEQ ID NO: 134 and 135)(IRESprobeF1, R1). We selected the ES clones that showed a single 16 kbband in the Southern blotting. The selected ES clones were also testedby karyotype analysis according to the method described in ShinichiAizawa, “Biomanual Series 8, Gene Targeting”, published by Yodosha,1995. One ES clone, #2, that showed normal karyotype were used forinjection into embryos.

IRESprobeF1 (SEQ ID NO: 134): CTAACGTTACTGGCCGAAGC IRESprobeR1 (SEQ IDNO: 135): ATTATCATCGTGTTTTTCAAAGGAA

The cells in a frozen stock of the transfected ES cell clones #2 werethawed, started to culture and injected into 8 cell stage embryosobtained by mating a male and a female mouse of MCH(ICR) mouse strain(CREA JAPAN, INC.); the injection rate was 10-12 cells per embryo. Afterthe embryos were cultured overnight in the medium for ES cells todevelop into blastocysts, about ten of the ES cell-injected embryos weretransplanted to each side of the uterus of a foster mother ICR mouse(CREA JAPAN, INC.), which had been subjected to a pseudopregnanttreatment for 2.5 days. Contribution of the TT2F (agouti) ESclone-derived tissues in host embryo (albino) derived tissues can bedetermined eye pigmentation in embryos and coat color in viableoffspring.

(17) Expression of Human GIPF-GFP mRNA in Transgenic Chimaeric Mice

Total RNA samples were prepared from intestinal tract of pPv01GFP/TT2F-#2 derived chimaeras at various developmental stages (E13.5,E16.5, E19.5, day 3, day 7) and were subjected to semi-quantitativeRT-PCR analysis to examine GIPF-GFP mRNA expression. First-strand cDNAwas synthesized with Superscript III (Invitrogen) using random hexamersand 500 ng of total RNA extracted from the intestinal tract ofpPv01GFP/TT2F-#2 derived chimaeras and control TT2F-derived chimaeras byusing Isogen (Nippon Gene) and RNasy Mini (QIAGEN). Semi-quantitativeRT-PCR analysis was carried out using the cDNA at specific annealingtemperatures for each primer pair. PCR products were electrophoresed on2% agar gels and stained with ethidium bromide. The integrity of RNA wascontrolled by the amplification of cDNA generated by the murine GAPDH.The nucleotide sequences and annealing temperature of primer sets forGIPF (Pv01RT F1, R1; SEQ ID NO: 136 and 137), Axin 2 (Axin2 F, R; SEQ IDNO: 138 and 139)) and mGAPDH (mGAP DH5, 3; SEQ ID NO: 140 and 141) arelisted below.

Pv01RT F1 (SEQ ID NO: 136) GCTCTGACAGCAAGGAGACG Pv01RT R1 (60° C.) (SEQID NO: 137) CCCTAGGAATGCTCGTCAAG Axin2 F(MUS) (SEQ ID NO: 138)CAGGAGCCTCACCCTTCG Axin2 R(MUS) (60° C.) (SEQ ID NO: 139)ACGCCGAGGTGCTTGCCC mGAPDH5 (SEQ ID NO: 140) CACCATGGAGAAGGCCGGGGCCCACmGAPDH3 (65° C.) (SEQ ID NO: 141) ATCATACTTGGCAGGTTTCTCCAGG

As shown in FIG. 55, the GIPF-GFP transcripts were detectable at E13.5in intestinal tract of pPv01GFP/TT2F-#2 derived chimaeras and notdetected in in all the liver samples examined, which is well consistentwith the previous study (Madison et al., J. Biol. Chem. 277, p33275-33283, 2002) describing that expression of transgene driven by 13kb villin promoter is first detectable in the embryonic hindgut andmidgut at 12.5 dpc., and the expression is largely specific forintestinal epithelium. It is also evident that the expression level ofGIPF-GFP transcripts is gradually elevated with age during the course ofdevelopment. Eek-hoon et al. reported that endogenous Axin2 mRNAexpression could be induced by activation of the Wnt signaling pathway(Mol. Cell. Biol. 22, 1172-1183, 2002). It is also known that the Wntsignaling play a critical role in the development of intestinal tract.We therefore examined Axin2 expression in intestinal tract ofpPv01GFP/TT2F-#2 derived chimaeras. The result (FIG. 55) shows that theelevated expression of Axin2 mRNA is apparent at day 3 and 7 whencompared to the control chimaeras, suggesting that the expression ofhuman GIPF results in activation Wnt signaling pathway in intestinaltract of newborn.

(18) Stabilization of β-Catenin in Intestinal Tract of TransgenicChimaeric Mice

To evaluate the effect of GIPF on the Wnt/β-catenin signaling pathway,the stabilization of β-catenin was measured in small intestine and colonsampled from pPv01GFP/TT2F-#2 derived chimaeras at day 10. The procedurefor β-catenin stabilization assay is described in Example 17. As shownin FIG. 56, the expression of GIPF strongly induced the stabilization ofβ-catenin in both small intestine and colon sampled frompPv01GFP/TT2F-#2 (Pv01#2) derived chimaeras when compared to the controlchimaeras (wild-type).

(19) Evaluation of Phenotypic Changes in Intestinal Tract of TransgenicChimaeric Mice

Newborn pPv01GFP/TT2F-#2 derived chimaeric pups showed a significantabdominal distention at day 3 and the extent of this phenotype graduallyintensified with age. Visual inspection of day 3 chimaeras at necropsyshowed a remarkable enlargement in diameter throughout the smallintestine, associated with augmented surface vascularisation. Wholeembryos, pups or gastrointestinal tracts were fixed in Bouin solution.Paraffin embedded sections were stained with hematoxiyline and eosin(H&E) for histological evaluation. As shown FIG. 57, histopathologicalanalysis of H&E sections from pPv01GFP/TT2F-#2 (Pv01#2) revealedincrease in number of crypts and branching from embryonic day 19.5 (E19.5) to day 14 (d14) compared to control.

Example 31 Transgenic Chimaeric Mice that Express GIPF and Wnt3a inIntestinal Epithelial Cells (1) Preparation of Wnt3a Fragment (FIG. 58A)

Wnt3aFW (SEQ ID NO: 142) □CGGGATCCCCATGGCTCCTCTGGGATACCTCTTAGTGCTWnt3aRV (SEQ ID NO: 143) □GCTCTAGAGTTTAAACCTACTTGCAGGTGTGCACGTCATAG

KOD-puls-(TOYOBO) was used for the PCR reaction. The PCR reactionmixture contained 10 pmole of each primer (SEQ ID NO: 142 and 143) andthe human Wnt3a cDNA as a template. This PCR amplification was performedusing an initial denaturing incubation at 94° C. for three minutes. Then30 cycles of denaturation, annealing and amplification were performed byincubation at 94° C. for 15 sec and 68° C. for two minutes. A PCRproduct (approximately 1.06 kb) was purified by 0.8% agarose gelelectrophoresis and QIAquick Gel Extraction Kit (QIAGEN). Following thedigestion of isolated PCR product with BamHI and EcoRV, it was ligatedto pBluescriptIISK(−) that was digested with BamHI and XbaI, and treatedwith calf intestine alkaline phosphatase to dephosphorylate its bothends. The ligation mixture was transfected to DH5α and the DNA samplesprepared from the resultant transformants were analyzed by nucleotidesequencing to confirm the structure of inserted fragment. The cloneincluding a fragment with a correct nucleotide sequence was digestedwith NcoI and PmeI, and the fragment including the Wnt3a cDNA waspurified by electrophoresis using 0.8% agar and QIAquick Gel ExtractionKit (QIAGEN).

(2) Preparation of IRES/Wnt3a+pA Fragment (FIG. 58B)

The pIRES/GFP+pA plasmid DNA [see Example 30-(13)] was digested withNcoI and PmeI, and the vector fragment including without the GFP codingsequence was purified by 0.8% agarose gel electrophoresis. The fragmentincluding the Wnt3a cDNA [see (1)] was ligated to purified vectorfragment that was treated with E. coli C₇₋₅ alkaline phosphatase todephosphorylate its both ends. The ligation mixture was transfected toDH5α and the DNA samples prepared from the resultant transformants wereanalyzed by nucleotide sequencing to confirm the structure of insertedfragment. The clone including a fragment with a correct nucleotidesequence (pIRES/Wnt3a+pA) was digested with AscI and XhoI, and the DNAfragment including the IRES/Wnt3a+pA unit was purified by 0.8% agarosegel electrophoresis and QIAquick Gel Extraction Kit (QIAGEN).

(3) Construction of pPv01Wnt3a (FIG. 58C)

The pPv01GFP plasmid DNA [see Example 30-(13)] was digested withrestriction enzymes, AscI and XhoI, and the digested reaction mixturewas subjected to 0.8% agarose gel electrophoresis. The vector fragmentwithout the IRES/GFP+pA region was isolated and treated with calfintestine alkaline phosphatase to dephosphorylate its both ends. Theligation mixture of IRES/Wnt3a+pA fragment [see (2)] and the abovevector fragment was transfected to XL10-Gold Ultracompetent cells(STRATAGENE). The DNA samples prepared from the resultant transformantswere analyzed by nucleotide sequencing to confirm the structure ofinserted fragment. The clone including a fragment with a correctnucleotide sequence was selected (pPv01Wnt3a).

(4) Preparation of pPv01Wnt3a Plasmid DNA for Electroporation to MouseES Cells

The plasmid DNA of pPv01Wnt3a (60 μg) was digested with ClaI in thereaction mixture containing 1 mM spermidine (pH7.0, Sigma) for 5 hoursat 37° C. The reaction mixture was then subjected to phenol/chloroformextraction and ethanol precipitation (0.3M NaHCO₃) for 16 hours at −20°C. The linearized vector fragment was dissolved in HBS buffer (0.5μg/ul) and used for the following electroporation experiments.

(5) Production of Transgenic Chimaeric Mice Expressing Human GIPF andWnt3a in Intestinal Epithelial Cells

General procedures for obtaining mouse embryos, cultivation, injectionof the ES cells into the embryos, transplantation to the uteri of fostermothers were carried out in accordance with the method described inShinichi Aizawa, “Biomanual Series 8, Gene Targeting”, published byYodosha, 1995.

The linearized pPv01Wnt3a vector was transfect into C57BL/6×CBA F1strain derived mouse TT2F ES cells ((Uchida, 1995), Lifetech oriental)by electroporation according to the method described by Shinichi Aizawa,“Biomanual Series 8, Gene Targeting”, published by Yodosha, 1995. Theelectroporated ES cells were suspended in 20 ml of ES medium andinoculated into two 100 mm tissue culture plastic plates (Corning) intowhich feeder cells were seeded in advance. After one day, the medium wasreplaced with a medium containing a 200 μg/ml of G418 (Invitrogen).Seven to nine days thereafter, a total of 24 colonies for each vectorwere picked up. Each colonies was grown up to confluence in a 12-wellplate, and then four fifth of the culture was suspended in 0.2 ml ofcryopreservation medium (ES medium+10% DMSO (Sigma)) and stored frozenat −80° C. The remaining one fifth was inoculated into a 12-well gelatincoated plate and cultured for 2 days. Then, genomic DNA was isolatedusing the Puregene DNA Isolation Kit (Gentra System). Genomic DNAisolated from G418 resistant TT2F cells was digested with restrictionenzymes EcoRI and XhoI and then subjected to 0.8% agarose gelelectrophoresis. Using EcoRI-XhoI digestion, retention of an intactexpression unit including Villin promoter, human GIPF cDNA, Wnt3a cDNAand BGH polyA sequences of pPv01Wnt3a in the G418-resistant clones canbe determined by the detection of an approximately 16 kb band. SeparatedDNA fragments were transferred to a membrane (Gene Screen, NEN Lifescience Products) and then hybridization was carried out using the DNAfragment as probe prepared from IRES region of pPV01GFP [see Example30-(13)] by PCR using a primer set described in Example 30-(16)(IRESprobeF1, R1). We selected the clones that showed a single 16 kbband in the Southern blotting. The selected ES clones were also testedby karyotype analysis according to the method described in ShinichiAizawa, “Biomanual Series 8, Gene Targeting”, published by Yodosha,1995. Two ES clone, #7 and #13 that showed normal karyotype were usedfor injection into embryos.

The cells in a frozen stock of the transfected ES cell clones #7 and #13were thawed, started to culture and injected into 8 cell stage embryosobtained by mating a male and a female mouse of MCH(ICR) mouse strain(CREA JAPAN, INC.); the injection rate was 10-12 cells per embryo. Afterthe embryos were cultured overnight in the medium for ES cells todevelop into blastocysts, about ten of the ES cell-injected embryos weretransplanted to each side of the uterus of a foster mother ICR mouse(CREA JAPAN, INC.), which had been subjected to a pseudopregnanttreatment for 2.5 days. Contribution of the TT2F (agouti) ESclone-derived tissues in host embryo (albino) derived tissues can bedetermined eye pigmentation in embryos and coat color in viableoffspring.

(6) Expression of Human GIPF/Wnt3a mRNA in Transgenic Chimaeric Mice

Total RNA samples were prepared from intestinal tract ofpPv01Wnt3a/TT2F-#7, and #13 derived newborn chimaeras and were subjectedto semi-quantitative RT-PCR analysis to examine GIPF-GFP mRNAexpression. First-strand cDNA was synthesized with Superscript III(Invitrogen) using random hexamers and 500 ng of total RNA extractedfrom the intestinal tract of pPv01Wnt3a/TT2F-#7, and #13 derivedchimaeras and control TT2F-derived chimaeras by using Isogen (NipponGene) and RNasy Mini (QIAGEN). Semi-quantitative RT-PCR analysis wascarried out using the cDNA at specific annealing temperatures for eachprimer pair. PCR products were electrophoresed on 2% agar gels andstained with ethidium bromide. The integrity of RNA was controlled bythe amplification of cDNA generated by the murine GAPDH. The nucleotidesequences and annealing temperature of primer sets for GIPF (Pv01RT F1,R1), Axin 2 (Axin2 F, R) and mGAPDH (mGAPDH5, 3) are listed below.

Pv01RT F1 (SEQ ID NO: 136) GCTCTGACAGCAAGGAGACG Pv01RT R1 (60° C.) (SEQID NO: 137) CCCTAGGAATGCTCGTCAAG Axin2 F(MUS) (SEQ ID NO: 138)CAGGAGCCTCACCCTTCG Axin2 R(MUS) (60° C.) (SEQ ID NO: 139)ACGCCGAGGTGCTTGCCC mGAPDH5 (SEQ ID NO: 140) CACCATGGAGAAGGCCGGGGCCCACmGAPDH3 (65° C.) (SEQ ID NO: 141) ATCATACTTGGCAGGTTTCTCCAGG

As shown in FIG. 59, the GIPF-Wnt3a transcripts were detectable inintestinal tissues of pPv01Wnt3a/TT2F-#7 and -#13 derived from newbornchimaeras (Pv01Wnt3a: 1 to 4). The result (FIG. 59) also shows that theelevated expression of Axin2 mRNA is apparent when compared to thecontrol chimaeras (TT2F: 5 and 6).

(7) Stabilization of β-Catenin in Intestinal Tract of TransgenicChimaeric Mice

To evaluate the effect of GIPF on the Wnt/β-catenin signaling pathway,the stabilization of β-catenin was measured in small intestine and colonsampled from pPv01Wnt3a/TT2F-#7 and -#13 derived chimaeras. Theprocedure for β-catenin stabilization assay is described in Example 17.As shown in FIG. 60, the co-expression of GIPF and Wnt3a induced thestabilization of β-catenin in duodenum and colon sampled frompPv01Wnt3a/TT2F-#7 embryo (E20.5: 1 and 2) when compared to the controlchimaeras (wild-type: 3 and 4).

(8) Evaluation of Phenotypic Changes in Intestinal Tract of TransgenicChimaeric Mice

Newborn pPv01Wnt3a/TT2F-#7 and -#13 derived chimaeric pups showed asignificant abdominal distention. Visual inspection of newborn chimaerasat necropsy showed a remarkable enlargement in diameter throughout thesmall intestine, associated with augmented surface vascularisation.Whole embryos, pups or gastrointestinal tracts were fixed in Bouinsolution. Paraffin embedded sections were stained with hematoxiyline andeosin (H&E) for histological evaluation. As shown FIG. 61,histopathological analysis of H&E sections from pPv01Wnt3a/TT2F-#13embryonic day 20.5 embryo (E 20.5) revealed increase in number ofvillous cells, irregular branching and hyperplasia of villi. The extentof these phenotypes were stronger than those of pPv01GFP/TT2F-#2 derivedchimaeras [Example 30-(19)], suggesting the enhancement of GIPF actionby Wnt3a expression.

Example 32 RS-KO Mouse ES Cells A. Construction of the RS-KO Vector

The construction of the RS-KO vector (FIG. 62A) was performed accordingto the method described below, and depicted in FIGS. 62B-1K.

FIG. 62B Addition of the new restriction sites (NruI, SgrAI, and AscI)to pBluescript II SK(−) (Stratagene).

The oligo DNA fragments (SEQ ID NO: 144 and 145) for the addition of thenew restriction sites in pBluescript II SK(−) were synthesized.

(SEQ ID NO: 144) LinkA1:TCGAGTCGCGACACCGGCGGGCGCGCCC (SEQ ID NO: 145)LinkA2:TCGAGGGCGCGCCCGCCGGTGTCGCGAC

-   The prepared LinkA1 and LinkA2 were ligated into pBluescript II    SK(−) that was pre-digested with the restriction enzymes SalI and    XhoI. The resulting plasmid pBlueLA contained the newly added    restriction sites (NruI, SgrAI, and AscI).

FIG. 62C Addition of the New Restriction Sites (PacI, FseI, and SalI) topBlueLA.

The oligo DNA fragments (SEQ ID NO: 146 and 147) for the addition of thenew restriction sites in pBlueLA were synthesized.

SEQ ID NO: 146) LinkB1:GGCCGCTTAATTAAGGCCGGCCGTCGACG□ SEQ ID NO: 147)LinkB2:AATTCGTCGACGGCCGGCCTTAATTAAGC□

-   The prepared LinkB1 and LinkB2 were ligated into pBlueLA that was    pre-digested with the restriction enzymes NotI and EcoRI. The    resulting plasmid pBlueLAB contained the newly added restriction    sites (PacI, FseI, and SalI).

FIG. 62D Preparation of LoxP-Neo-B Fragment

-   LoxP-Neo-B fragment was prepared by T4 DNA polymerase treatment of    LoxP-Neo that was obtained from Xho/digestion of pLoxP-STneo (WO    00/10383).

FIG. 62E Preparation of pBlueLAB-LoxP-Neo Plasmid

LoxP-Neo-B fragment was ligated into pBlueLAB that was pre-digested withthe restriction enzyme EcoRV. The resulting plasmid pBlueLAB-LoxP-Neocontained LoxP-Neo-B fragment.

FIG. 62F Preparation of DT-A Fragment

pMC1 DT-A□GIBCO BRL□ was digested with XhoI and SalI, and the resultingDT-A fragment was separated and recovered from agarose gelelectrophoresis.

FIG. 62G Preparation of pBlueLAB-LoxP-Neo-DT-A Plasmid

DT-A fragment was ligated into pBlueLAB-LoxP-Neo that was pre-digestedwith the restriction enzyme XhoI. The resulting plasmidpBlueLAB-LoxP-Neo-DT-A contained DT-A fragment.

FIG. 62H Preparation of 3′Genomic Region of RS Element

The forward (RS3′FW2; SEQ ID NO: 148) and reverse (RS3′RV3; SEQ ID NO:149) primers for PCR were synthesized based on the sequence of the mouseobtained from GenBank (Accession Number□AC090291), and used to amplifythe DNA of 3′ genomic region of RS element.RS3′FW2: TTGGCGCGCCCTCCCTAGGACTGCAGTTGAGCTCAGATTTGA (SEQ ID NO: 148) wasprepared by adding a AscI recognition sequence at 5′ end site, andRS3′RV3: CCGCTCGAGTCTTACTGTCTCAGCAACAATAATATAAACAGGGG (SEQ ID NO: 149)was prepared by adding a XhoI recognition sequence at 5′ end site. PCRwas carried out using BAC clone RP23-43514 (GenBank Accession NumberAC090291) as template. The PCR product was digested with restrictionenzymes AscI and XhoI, and ligated into pBlueLAB that was pre-digestedwith the restriction enzymes AscI and XhoI. The resulting plasmidcontained the designated DNA sequence of 3′genomic region of RS elementwith no substitution in nucleotide sequence within the region betweenAscI and XhoI was treated with AscI and XhoI and then the 3′genomicregion of RS element (about 2 Kb) was obtained.

FIG. 62I Insertion of 3′Genomic Region of RS Element intopBlueLAB-LoxP-Neo-DT-A

The 3′genomic region of RS element was ligated intopBlueLAB-LoxP-Neo-DT-A that was pre-digested with AscI and XhoI. Afterverifying the connecting regions between pBlueLAB-LoxP-Neo-DT-A and the3′genomic region of RS element, the plasmid pBlueLAB-LoxP-Neo-DT-A-3′RSwas obtained.

FIG. 62J Preparation of 5′Genomic Region of Mouse RS Element

The forward (RS5′FW3; SEQ ID NO: 150) and the reverse (RS5′RV3; SEQ IDNO: 151) primers for PCR were synthesized based on the sequence of themouse obtained from GenBank (Accession Number AC090291), and used toamplify the DNA of 5′genomic region of RS element.RS5′FW3: ATAAGTGCGGCCGCAAAGCTGGTGGGTTAAGACTATCTCGTGAAGTG (SEQ ID NO:150) was prepared by adding a NotI recognition sequence at 5′ end site,and RS5′RV3: ACGCGTCGACTCACAGGTTGGTCCCTCTCTGTGTGTGGTTGCTGT (SEQ ID NO:151) was prepared by adding a SalI recognition sequence at 5′ end site.PCR was carried out using BAC clone RP23-43514 (GenBank Accession NumberAC090291) as template. The PCR product was digested with restrictionenzymes NotI and SalI, and ligated into pBlueLAB that was pre-digestedwith the restriction enzymes NotI and SalI. The resulting plasmidcontained the designated DNA sequence of 5′genomic region of RS elementwith no substitution in nucleotide sequence within the region betweenNotI and SalI was treated with NotI and SalI and then the 5′genomicregion of RS element (about 5 Kb) was obtained.

FIG. 62K Insertion of 5′Genomic Region of RS Element intopBlueLAB-LoxP-Neo-DT-A-3′RS

The 5′genomic region of RS element was ligated intopBlueLAB-LoxP-Neo-DT-A-3′RS that was pre-digested with NotI and SalI.After verifying the connecting regions betweenpBlueLAB-LoxP-Neo-DT-A-3′RS and the 5′genomic region of RS element, theRS-KO vector was constructed.

B. Preparation of RS-KO Mouse ES Cells

General procedures for obtaining mouse embryos and cultivation werecarried out in accordance with the method described in Aizawa Shinichi,“Biomanual Series 8, Gene Targeting”, published by Yodosha, 1995. TheRS-KO vector was linearized with Not I and transferred into C57BL/6×CBAF1 derived mouse TT2F ES cells ((Uchida, 1995), Lifetech oriental) byelectroporation according to the method described by Shinichi Aizawa,“Biomanual Series 8, Gene Targeting”, published by Yodosha, 1995. Theelectroporated ES cells were suspended in 20 ml of ES medium [DMEM(GIBCO), 18% FBS (GIBCO), 0.1 mM 2-mercaptoethanol (GIBCO), 1000 U/mlLIF (leukemia inhibitory factor, CHEMICON International, Inc.)] andinoculated into two 100 mm tissue culture plastic plates (Corning) intowhich feeder cells (Invitrogen) were seeded in advance. After one day,the medium was replaced with a medium containing 0.75 g/ml of puromycin(Sigma). Seven days thereafter, puromycin resistant colonies formed werepicked up. Each colony was grown up to confluence in a 24-well plate,and then two third of the culture was suspended in 0.2 ml ofcryopreservation medium [FBS+10% DMSO (Sigma)] and stored frozen at −80°C. The remaining one third was inoculated into a 12-well gelatin coatedplate and cultured for 2 days. Then, genomic DNA was isolated using thePuregene DNA Isolation Kit (Gentra System).

3′KO-probe for Southern analysis was prepared as follows. RS3′ SouthernFW1 (SEQ ID NO: 152) and RS3′ Southern RV2 (SEQ ID NO: 153) primers weresynthesized based on the sequence of the mouse obtained from GenBank(Accession Number AC090291), and used to amplify about 600 mer long DNAfragment of 3′ genomic region of RS element.

SEQ ID NO: 152) RS3′Southern FW1: TCTTACTAGAGTTCTCACTAGCTCT□ SEQ ID NO:153) RS3′Southern RV2: GGAACCAAAGAATGAGGAAGCTGTT□

Genomic DNA isolated from puromycin resistant TT2F cells was digestedwith restriction enzyme EcoR I (Takara Shuzo) and then subjected to 0.8%agarose gel electrophoresis.

Separated DNA fragments were transferred to a membrane (GeneScreen, NEN™Life Science Products) and then hybridization was carried out using theDNA fragment as probe prepared from 3′ genomic region of RS element DNA(3′KO-probe). The band pattern of untargeted ES clone shows one band ofMW of about 5.7 Kb and targeted ES clone shows two bands of MW of about5.7 Kb and 7.4 Kb (FIG. 63). The selected RS-KO mouse ES clones werealso tested by karyotype analysis according to the method described byShinichi Aizawa, “Biomanual Series 8, Gene Targeting”, published byYodosha, 1995. The RS-KO mouse ES clones that showed normal karyotypewere used for further experiments.

Example 33 Transgenic GIPF Deletion Mutant Animals A. Construction ofthe pCk m4 KI Vector.

The construction of the GIPF deletion mutant 4 Ck knock-in (pCk m4 KI)vector (FIG. 64A) was performed according to the method described below,and depicted in FIGS. 64B-64K.

FIG. 64B Preparation of Ck P2 KI+AS KI

The oligo DNA fragments (SEQ ID NO: 154 and 155) for the addition of thenew restriction sites in Ck P2 KI were synthesized.

(SEQ ID NO: 154) Ascl top linker: GGCCAGGCGCGCCTTGC (SEQ ID NO: 155)Ascl bottom linker: GGCCGCAAGGCGCGCCTThe prepared AscI top linker and AscI bottom linker were ligated into CkP2 KI that was pre-digested with the restriction enzyme NotI. Theresulting plasmid Ck P2 KI+AS KI contained the newly added restrictionsite (AscI).

FIG. 64C Preparation of pBlueLAB+Nh

The oligo DNA fragments (SEQ ID NO: 156 and 157) for the addition of thenew restriction sites in pBlueLAB were synthesized.

(SEQ ID NO: 156) Pac-Nhe-Fse S: TAAGGGCTAGCTAGGGCCGG (SEQ ID NO: 157)Pac-Nhe-Fse AS: CCCTAGCTAGCCCTTAATThe prepared Pac-Nhe-Fse S and Pac-Nhe-Fse AS were ligated into pBlueLABthat was pre-digested with the restriction enzymes PacI and FseI. Theresulting plasmid pBlueLAB+Nh contained the newly added restriction site(NheI).

FIG. 64D Preparation of pBlueLAB+NhHp

The oligo DNA fragments (SEQ ID NO: 158 and 159) for the addition of thenew restriction sites in pBlueLAB+Nh were synthesized.

S/HpaI/Hd-S: TCGAGTTAAC (SEQ ID NO: 158) S/HpaI/Hd-AS: AGCTGTTAAC (SEQID NO: 159)

-   The prepared S/HpaI/Hd-S and S/HpaI/Hd-AS were ligated into    pBlueLAB+Nh that was pre-digested with the restriction enzymes SalI    and HindIII. The resulting plasmid pBlueLAB+NhHp contained the newly    added restriction site (HpaI).

FIG. 64E Preparation of pCkpAP2

pCkP2+As KI was digested with HpaI and NheI, and the resulting 952 bpfragment was separated and recovered from agarose gel electrophoresis.The 952 bp fragment was ligated into pBlueLAB+NhHp that was pre-digestedwith the restriction enzymes HpaI and NheI. The resulting plasmidpCkpAP2 contained the 952 bp fragment.

FIG. 64F Preparation of pCkpAMCS

The oligo DNA fragments (SEQ ID NO: 160 and 161) for the addition of thenew restriction sites in pCkpAP2 were synthesized.

(SEQ ID NO: 160) SPFNlinker-S: AGCTGTCGACTTAATTAAGGCCGGCCG (SEQ ID NO:161) SPFNlinker-AS: CTAGCGGCCGGCCTTAATTAAGTCGAC

-   The prepared SPFNlinker-S and SPFNlinker-AS were ligated into    CkpAMCS that was pre-digested with the restriction enzymes HindIII    and NheI. The resulting plasmid CkpAMCS contained the newly added    restriction site (PacI).

FIG. 64G Preparation of pCkP2ΔP

pCkpAMCS was digested with HpaI and NheI, and the resulting about 700 bplong fragment was separated and recovered from agarose gelelectrophoresis. The 700 bp fragment was ligated into pCkP2+As KI thatwas pre-digested with the restriction enzymes HpaI and NheI. Theresulting plasmid pCkP2ΔP contained the 700 bp fragment.

FIG. 64H Preparation of pBS+PFN

The oligo DNA fragments (SEQ ID NO: 162 and 163) for the addition of thenew restriction sites in pBluescript II SK (−) were synthesized.

(SEQ ID NO: 162) S/PFN/Hd-S: TCGACTTAATTAAGGCCGGCCCTAGCTAGCA (SEQ ID NO:163) S/PFN/Hd-AS: AGCTTGCTAGCTAGGGCCGGCCTTAATTAAGThe prepared S/PFN/Hd-S and S/PFN/Hd-AS were ligated into pBluescript IISK(−) that was pre-digested with the restriction enzymes SalI andHindIII. The resulting plasmid pBSnPFN contained the newly addedrestriction sites (PacI, FseI, and NheI).

FIG. 64I Preparation of pPSs3.8

The forward (PsecSP FW1; SEQ ID NO: 164) and reverse (PsecSP RV; SEQ IDNO: 165) primers for PCR were synthesized based on the sequence of themouse obtained from GenBank (Accession Number K02159), and used toamplify the DNA of promoter and leader sequence coding region of IgK.The leader sequence coding region contained intrinsic intron sequence.PsecSP FW1: CCTTAATTAAAGTTATGTGTCCTAGAGGGCTGCAAACTCAAGATC (SEQ ID NO:164) was prepared by adding a PacI recognition sequence at 5′ end site,and PsecSP RV: TTGGCCGGCCTTGGCGCCAGTGGAACCTGGAATGATAAACACAAAGATTATTG(SEQ ID NO: 165) was prepared by adding a FseI recognition sequence atSend site. PCR was carried out using the mouse genome from TT2F ES cells((Uchida, 1995), Lifetech oriental) as template. The PCR product wasdigested with restriction enzymes PacI and FseI, and ligated intopBS□PFN that was pre-digested with the restriction enzymes PacI andFseI. The resulting plasmid pPSs3.8 contained the DNA fragment ofpromoter and leader sequence coding region of IgK.

FIG. 64J Preparation of m4(+SP) and m4(−SP)

The forward (Sal kozak GIPF F; SEQ ID NO: 166) and reverse (GIPF m4 RVFse; SEQ ID NO: 167) primers for PCR were synthesized based on thesequence of the deletion mutant #4 (FIG. 43; SEQ ID NO: 91) and used toamplify the DNA of leader sequence of GIPF and deletion mutant #4sequence of GIPF.Sal kozak GIPF F: MG CGT CGA CCA CCA TGC GGC TTG GGC TGT GTG (SEQ ID NO:166) was prepared by adding a SalI recognition sequence at 5′ end site,and GIPF m4 RV Fse: ATG GCC GGC CCT ACA TGG TGC CAT TGG CAG (SEQ ID NO:167) was prepared by adding a FseI recognition sequence at 5′ end site.PCR was carried out using the GIPF KI vector as template. The PCRproduct was digested with restriction enzymes SalI and FseI, and thenthe m4(+SP) fragment was obtained.

The forward (Hy01(−SP) FW; SEQ ID NO: 168) and reverse (GIPF m4 RV Fse;SEQ ID NO:169) primers for PCR were synthesized based on the sequence ofthe deletion mutant #4 (FIG. 43; SEQ ID NO: 91) and used to amplify theDNA of deletion mutant#4 sequence of GIPF.

Hy01(−SP) FW: AGCCGGGGGATCAAGGGGAAAAGGCAGAGG (SEQ ID NO: 168) wasprepared by adding a phosphoric acid at 5′ end site, and GIPF m4 RV Fse:ATG GCC GGC CCT ACA TGG TGC CAT TGG CAG (SEQ ID NO: 169) was prepared byadding a FseI recognition sequence at 5′ end site. PCR was carried outusing the GIPF KI vector as template. The PCR product was digested withrestriction enzyme FseI, and then the m4(−SP) fragment was obtained.

FIG. 64K Construction of pCk m4 KI Vector

The m4(+SP) fragment was ligated into pCkP2+As KI that was pre-digestedwith the restriction enzymes SalI and FseI. The resulting pCk m4 KIvector contained the m4(+SP) fragment, that was used for the generationof the GIPF deletion mutant mice.B. Construction of the pPSm4 KI Vector.The construction of the GIPF deletion mutant 4 PS knock-in (pPS m4 KI)vector (FIG. 65A) was performed according to the method described below,and depicted in FIGS. 65B-65C.

FIG. 65B Preparation of pPSs3.8 m4

The m4(−SP) fragment was ligated into pPSs3.8 that was pre-digested withthe restriction enzymes SfoI and FseI. The resulting pPSs3.8 m4contained the m4(−SP) fragment.

FIG. 65C Construction of pPSm4 KI Vector

pPSs3.8 m4 was digested with PacI and FseI, and the resulting about 1.2Kb long fragment was separated and recovered from agarose gelelectrophoresis. The 1.2 Kb fragment was ligated into CkP2ΔP that waspre-digested with the restriction enzymes PacI and FseI. The resultingpPSm4 KI vector contained the 1.2 Kb fragment, that was used for thegeneration of the GIPF deletion mutant mice.

C. Generation of GIPF Deletion Mutant Mice

General procedures for obtaining mouse embryos, cultivation, injectionof the ES cells into the embryos, transplantation to the uteri of fostermothers were carried out in accordance with the method described inAizawa Shinichi, “Biomanual Series 8, Gene Targeting”, published byYodosha, 1995.

The pCkm4 KI vector and pPSm4 KI vector were linearized with NotI andtransferred into RS-KO mouse ES cells by electroporation according tothe method described by Shinichi Aizawa, “Biomanual Series 8, GeneTargeting”, published by Yodosha, 1995. The electroporated RS-KO mouseES cells were suspended in 20 ml of ES medium [DMEM (GIBCO), 18% FBS(GIBCO), 0.1 mM 2-mercaptoethanol (GIBCO), 1000 U/ml LIF (leukemiainhibitory factor, CHEMICON International, Inc.)] and inoculated intotwo 100 mm tissue culture plastic plates (Corning) into which feedercells (Invitrogen) were seeded in advance. After one day, the medium wasreplaced with a medium containing 0.75 g/ml of puromycin (Sigma). Sevento nine days thereafter, 24 colonies formed from pCkm4 KI vectorelectroporated RS-KO mouse ES cells and 24 colonies formed from pPSm4 KIvector electroporated RS-KO mouse ES cells were picked up, respectively.Each colony was grown up to confluence in a 12-well plate, and then twothird of the culture was suspended in 0.2 ml of cryopreservation medium[FBS+10% DMSO (Sigma)] and stored frozen at −80° C. The remaining onethird was inoculated into a 12-well gelatin coated plate and culturedfor 2 days. Then, genomic DNA was isolated using the Puregene DNAIsolation Kit (Gentra System). Genomic DNA isolated from puromycinresistant RS-KO mouse ES cells was digested with restriction enzyme EcoRI (Takara Shuzo) and then subjected to 0.8% agarose gel electrophoresis.Separated DNA fragments were transferred to a membrane (GeneScreen, NEN™Life Science Products) and then hybridization was carried out using theDNA fragment as probe prepared from 3′ region of IgJκ-Cκgenomic DNA (Xho1-EcoR I, 1.3 Kb (SEQ ID NO: 67): WO 00/10383, Example No. 48). The bandpattern of untargeted ES clone shows one band of MW of 15.6 Kb. pCkm4 KItargeted RS-KO mouse ES clone shows two bands of MW of 15.6 Kb and 12.9Kb and pPSm4 KI targeted RS-KO mouse ES clone shows two bands of MW of15.6 Kb and 12.5 Kb, respectively (FIGS. 66 and 67). One out of 10 pCkm4KI targeted RS-KO mouse ES clones #3 was selected after Southernanalysis (rate of homologues recombination was about 41.7%) and one outof 7 pPSm4 KI targeted RS-KO mouse ES clone #7 was selected afterSouthern analysis (rate of homologues recombination was about 29.2%).The selected ES clones were also tested by karyotype analysis accordingto the method described by Shinichi Aizawa, “Biomanual Series 8, GeneTargeting”, published by Yodosha, 1995. One clone #3 of pCkm4 KItargeted RS-KO mouse ES clones and one clone #7 of pPSm4 KI targetedRS-KO mouse ES clones that showed normal karyotype were used forimplantation into embryos, respectively.

The cells in a frozen stock of the pCkm4 KI targeted RS-KO mouse ES cellclone #3 and the pPSm4 KI targeted RS-KO mouse ES cell clone #7 werethawed, started to culture and injected into 8-cell stage embryosobtained by mating a male and a female mouse of Immunoglobulin heavychain knock out mouse strain (Tomizuka et. Al. Proc. Natl. Acad. Sci.USA, 97: 722-727, 2000); the injection rate was 10-12 cells per embryo.After the embryos were cultured overnight in the medium for ES cells todevelop into blastocysts, about ten of the ES cell-injected embryos weretransplanted to each side of the uterus of a foster mother ICR mouse(CREA JAPAN, INC.), which had been subjected to a pseudopregnanttreatment for 2.5 days. As a result of transplantation of each of 80injected embryos, 15 and 20 offspring mice were born, respectively.Chimerism in the offspring was determined by the extent of TT2Fcell-derived agouti coat color (dark brown) in the host embryo(ICR)-derived albino coat color. Out of the 15 offspring, 9 mice (pCkm4knock-in mice) were recognized to have partial agouti coat color,indicating the contribution of the RS-KO mouse ES cells and out of the20 offspring, 16 mice (pPSm4 knock-in mice) were recognized to havepartial agouti coat color, indicating the contribution of the RS-KOmouse ES cells. GIPF deletion mutant mice were obtained same asdescribed above from the other clones of the pCkm4 KI targeted RS-KOmouse ES cells and the pPSm4 KI targeted RS-KO mouse ES cells.

Mice were kept under a 12/12-hour dark/light cycle (lights on at 8:00am) and received 5 μm filtered water and CE-2 food (CLEA JAPAN, INC.) adlibitum. Male mice were housed individually after weaning period.

Example 34 Transgenic GIPF Variant Animals A. Construction of the pCk VRKI Vector.

The construction of the GIPF variant Ck knock-in (pCk VR KI) vector(FIG. 68A) was performed according to the method described below, anddepicted in FIGS. 68B-68C.

FIG. 6B Preparation of GIPF variant with kozak (VR+kz) and GIPF variant(VR).

The forward (VR Ck Fw; SEQ ID NO: 171) and reverse (VR KI Rv; SEQ ID NO:171) primers for PCR were synthesized based on the sequence of GenBank(Accession Number AK098225), and used to amplify the DNA of GIPF variantwith kozak at 5′ end site.VR Ck Fw: ACG CGT CGA CCA CCA TGA TAT TCC GAG TCA GTG C (SEQ ID NO: 170)was prepared by adding a SalI recognition sequence at 5′ end site, andVR KI Rv: GGC CGG CCC TAG GCA GGC CCT GCA GAT GTG AGT GG (SEQ ID NO:171) was prepared by adding a FseI recognition sequence at 5′ end site.PCR was carried out using the GIPF KI vector as template. The PCRproduct was digested with restriction enzymes SalI and FseI, and thenVR+kz was obtained.The forward (VR Fw; SEQ ID NO: 172) and reverse (VR KI Rv; SEQ ID NO:173) primers for PCR were synthesized based on the sequence of GenBank(Accession Number AK098225), and used to amplify the DNA of GIPFvariant.VR Fw: ATG ATA TTC CGA GTC AGT GCC GAG GGG AGC CAG (SEQ ID NO: 172) wasprepared by adding a phosphoric acid at 5′ end site, and VR KI Rv: GGCCGG CCC TAG GCA GGC CCT GCA GAT GTG AGT GG (SEQ ID NO: 173) was preparedby adding a FseI recognition sequence at 5′ end site. PCR was carriedout using the GIPF KI vector as template. The PCR product was digestedwith restriction enzyme FseI, and then the VR was obtained.

FIG. 68C Construction of pCk VR KI Vector

The VR+kz was ligated into pCkP2+As KI that was pre-digested with therestriction enzymes SalI and FseI. The resulting pCk VR KI vectorcontained the VR+kz, that was used for the generation of the GIPFvariant mice.

B. Construction of the pPS VR KI Vector.

The construction of the GIPF variant PS knock-in (pPS VR KI) vector(FIG. 69A) was performed according to the method described below, anddepicted in FIGS. 69B-69C.

FIG. 69B Preparation of pPSs3.8VR

The VR was ligated into pPSs3.8 that was pre-digested with therestriction enzymes SfoI and FseI. The resulting pPSs3.8VR contained theVR.

FIG. 69C Construction of pPS VR KI Vector

pPSs3.8VR was digested with SalI and FseI, and the resulting about 1.5Kb long fragment was separated and recovered from agarose gelelectrophoresis. The 1.5 Kb fragment was ligated into □CkP2ΔP that waspre-digested with the restriction enzymes SalI and FseI. The resultingpPS VR KI vector contained the 1.5 Kb fragment, that was used for thegeneration of the GIPF variant mice.

C. Generation of GIPF Variant Mice

General procedures for obtaining mouse embryos, cultivation, injectionof the ES cells into the embryos, transplantation to the uteri of fostermothers were carried out in accordance with the method described inAizawa Shinichi, “Biomanual Series 8, Gene Targeting”, published byYodosha, 1995.

The pCkVR KI vector and pPSVR KI vector were linearized with Not I andtransferred into RS-KO mouse ES cells by electroporation according tothe method described by Shinichi Aizawa, “Biomanual Series 8, GeneTargeting”, published by Yodosha, 1995. The electroporated RS-KO mouseES cells were suspended in 20 ml of ES medium [DMEM (GIBCO), 18% FBS(GIBCO), 0.1 mM 2-mercaptoethanol (GIBCO), 1000 U/ml LIF (leukemiainhibitory factor, CHEMICON International, Inc.)] and inoculated intotwo 100 mm tissue culture plastic plates (Corning) into which feedercells (Invitrogen) were seeded in advance. After one day, the medium wasreplaced with a medium containing 0.75 g/ml of puromycin (Sigma). Sevento nine days thereafter, 24 colonies formed from pCkVR KI vectorelectroporated RS-KO mouse ES cells and 24 colonies formed from pPSVR KIvector electroporated RS-KO mouse ES cells were picked up, respectively.Each colony was grown up to confluence in a 12-well plate, and then twothird of the culture was suspended in 0.2 ml of cryopreservation medium[FBS+10% DMSO (Sigma)] and stored frozen at −80° C. The remaining onethird was inoculated into a 12-well gelatin coated plate and culturedfor 2 days. Then, genomic DNA was isolated using the Puregene DNAIsolation Kit (Gentra System). Genomic DNA isolated from puromycinresistant RS-KO mouse ES cells was digested with restriction enzyme EcoRI (Takara Shuzo) and then subjected to 0.8% agarose gel electrophoresis.Separated DNA fragments were transferred to a membrane (GeneScreen, NEN™Life Science Products) and then hybridization was carried out using theDNA fragment as probe prepared from 3′ region of IgJκ-Cκgenomic DNA (XhoI-EcoR I, 1.3 Kb (SEQ ID NO: 67): WO 00/10383, Example No. 48). The bandpattern of untargeted ES clone shows one band of MW of 15.6 Kb. pCkVR KItargeted RS-KO mouse ES clone shows two bands of MW of 15.6 Kb and 13.1Kb and pPSVR KI targeted RS-KO mouse ES clone shows two bands of MW of15.6 Kb and 12.9 Kb, respectively (FIGS. 70 and 71). One out of 12 pCkVRKI targeted RS-KO mouse ES clones #3 was selected after Southernanalysis (rate of homologues recombination was about 37.5%) and one outof 8 PPSVR KI targeted RS-KO mouse ES clone #14 was selected afterSouthern analysis (rate of homologues recombination was about 25%). Theselected ES clones were also tested by karyotype analysis according tothe method described by Shinichi Aizawa, “Biomanual Series 8, GeneTargeting”, published by Yodosha, 1995. One clone #3 of pCkVR KItargeted RS-KO mouse ES clones and one clone #14 of PPSVR KI targetedRS-KO mouse ES clones that showed normal karyotype were used forimplantation into embryos, respectively.

The cells in a frozen stock of the pCkVR KI targeted RS-KO mouse ES cellclone #3 and the pPSVR KI targeted RS-KO mouse ES cell clone #14 werethawed, started to culture and injected into 8-cell stage embryosobtained by mating a male and a female mouse of Immunoglobulin heavychain knock out mouse strain (Tomizuka et. Al. Proc. Natl. Acad. Sci.USA, 97: 722-727, 2000); the injection rate was 10-12 cells per embryo.After the embryos were cultured overnight in the medium for ES cells todevelop into blastocysts, about ten of the ES cell-injected embryos weretransplanted to each side of the uterus of a foster mother ICR mouse(CREA JAPAN, INC.), which had been subjected to a pseudopregnanttreatment for 2.5 days. As a result of transplantation of each of 220injected embryos, 68 and 60 offspring mice were born, respectively.Chimerism in the offspring was determined by the extent of TT2Fcell-derived agouti coat color (dark brown) in the host embryo(ICR)-derived albino coat color. Out of the 68 offspring, 47 mice (pCkVRknock-in mice) were recognized to have partial agouti coat color,indicating the contribution of the RS-KO mouse ES cells and out of the60 offspring, 38 mice (PPSVR knock-in mice) were recognized to havepartial agouti coat color, indicating the contribution of the RS-KOmouse ES cells. GIPF variant mice were obtained same as described abovefrom the other clones of the pCkVR KI targeted RS-KO mouse ES cells andthe pPSVR KI targeted RS-KO mouse ES cells.

Mice were kept under a 12/12-hour dark/light cycle (lights on at 8:00am) and received 5 μm filtered water and CE-2 food (CLEA JAPAN, INC.) adlibitum. Male mice were housed individually after weaning period.

Example 35 Evaluation of the Biological Activity of GIPF Deletion MutantUsing Transgenic GIPF Deletion Mutant-KI Mice

The gross pathological changes and the histological changes of the smallintestine and colon from the transgenic mice described above wereevaluated as follows. GIPF deletion mutant-KI (Ckm4-KI and PSm4-KI) micewere harvested at 4 weeks of age. Gross appearance of GIPF deletionmutant-KI gastrointestinal tract showed little difference compared tocontrol (FIG. 72). For histopathological evaluation, gastrointestinaltract were removed and fixed in formalin. Paraffin embedded sectionswere stained with hematoxiyline and eosin (H&E) for histologicalevaluation. H&E sections of small intestine were shown FIG. 73 (lowmagnification) and FIG. 74 (high magnification).

H&E sections from GIPF deletion mutant-KI mouse small intestine revealedmild increase of crypt length and number compared to control. Theincrease of crypt length and number in PSm4-KI was tended to be greaterthan Ckm4-KI.

Expression of GIPF deletion mutant #4 gene expression and the inductionof β-catenin targeted Axin-2 gene expression were analyzed using smallintestine and colon samples derived from GIPF deletion mutant-KI mice.50 mg of ileum, colon and liver samples were removed and rapidly frozeby use of liquid nitrogen. Frozen sections were homogenized with 1 ml ofISOGEN (NIPPON GENE) and total RNA was extracted under the recommendedconditions. To remove genomic DNA, the RNA solution was treated withDNase (WAKO; Deoxyribonuclease RT grade) at 37° C. for 15 mins. Thentotal RNA was purified with RNasy Mini (QIAGEN) under the recommendedconditions. For each sample, cDNA was synthesized from 500 ng of totalRNA using Super Script III (Invitrogen) under the recommendedconditions. Then cDNA was treated with 1 unit of RNaseH (Invitrogen) at37° C. for 20 mins to digest remaining RNA. PCR was carried out usingsynthesized cDNA as a template. The following two primers were useddetection of GIPF deletion mutant: PSm4RT F1: ATCAAGGGGAAAAGGCAGAG (SEQID NO: 174), and CkpolyA R2: CGCTTGTGGGGAAGCCTCCAAGACC (SEQ ID NO: 175).For detection of Axin-2, following two primers were used: Axin2 F (MUS):CAGGAGCCTCACCCTTCG (SEQ ID NO: 138), and Axin2 R (MUS):ACGCCGAGGTGCTTGCCC (SEQ ID NO: 139). Mouse GAPDH primers: mGAPDH5:CACCATGGAGAAGGCCGGGGCCCAC (SEQ ID NO: 140), and mGAPDH3:ATCATACTTGGCAGGTTTCTCCAGG (SEQ ID NO: 141) were used for the detectionof housekeeping gene expression. The reaction mixture of PCR wasprepared by adding sterilized distilled water to 2.5 ul of 10×LA PCRBuffer II (Takara Shuzo), 4 ul of 2.5 mM each dNTP Mixture (TakaraShuzo), 0.5 ul of 10 mM each primer, 2 ul of 10× cDNA diluted withsterilized distilled water and 0.5 ul of LA Taq (Takara Shuzo) to make25 ul. For detection of GIPF deletion mutant and Axin-2, reactionmixture of PCR was incubated at 94° C. for 2 mins, reaction of 33 cycleswas carried out, with 94° C. for 30 seconds, 60° C. for 30 seconds and72° C. for 30 seconds. For detection of GAPDH, reaction mixture of PCRwas incubated at 94° C. for 2 mins, reaction of 22 cycles was carriedout, with 94° C. for 30 seconds, 65° C. for 30 seconds and 72° C. for 30seconds.

GIPF deletion mutant gene expression was detected in ileum and colonfrom PSm4-KI mice. The expression level of Axin-2 was increased inPSm4-KI mice compared to control (FIG. 75). This data suggested thatdeletion mutant form of GIPF had activity to induce β-catenin targetedgene expression and responsible for activation of down stream ofβ-catenin signaling pathway.

Thus it suggested that GIPF deletion mutant #4 had activity to stimulateβ-catenin signaling pathway.

Example 36 Evaluation of the Biological Activity of GIPF Variant UsingTransgenic GIPF Variant-KI Mice

The gross pathological changes and the histological changes of the smallintestine and colon from the transgenic mice described above wereevaluated as follows. GIPF variant-KI (CkVR-KI and PSVR-KI) mice wereharvested at 4 weeks of age. Gross appearance of harvested PSVR-KIshowed that they had remarkable intestinal distension and increasedsmall intestinal mass when compared to control mouse (FIG. 76). Forhistopathological evaluation, gastrointestinal tract were removed andfixed in formalin. Paraffin embedded sections were stained withhematoxiyline and eosin (H&E) for histological evaluation. H&E sectionsof small intestine were shown FIG. 77 (low magnification) and FIG. 78(high magnification).

H&E sections from small intestine revealed remarkable difference betweenPSVR-KI and control. As shown FIG. 76, crypt cell hyperplasia of PSVR-KIdemonstrated increased in crypt length and number of Paneth cellscompared to control mouse. On the other hand, CkVR-KI did not showobvious intestinal distention or increase of crypt length (data notshown). The difference of observed phenotypes between CkVR-KI andPSVR-KI was estimated because of the difference of their KI-vectorstructure.

Expression of GIPF variant gene expression and the induction ofβ-catenin targeted Axin-2 gene expression were analyzed using smallintestine and colon samples derived from GIPF variant-KI mice. 50 mg ofileum, colon and liver samples were removed and rapidly froze by use ofliquid nitrogen. Frozen sections were homogenized with 1 ml of ISOGEN(NIPPON GENE) and total RNA was extracted under the recommendedconditions. To remove genomic DNA, the RNA solution was treated withDNase (WAKO; Deoxyribonuclease RT grade) at 37° C. for 15 mins. Thentotal RNA was purified with RNasy Mini (QIAGEN) under the recommendedconditions. For each sample, cDNA was synthesized from 500 ng of totalRNA using Super Script III (Invitrogen) under the recommendedconditions. Then cDNA was treated with 1 unit of RNaseH (Invitrogen) at37° C. for 20 mins to digest remaining RNA. PCR was carried out usingsynthesized cDNA as a template. The following two primers were useddetection of GIPF variant: PSFLJRT F1: ATAACTTCTGCACCAAGTGTAAGGA (SEQ IDNO: 176), and CkpolyA R2: CGCTTGTGGGGAAGCCTCCAAGACC (SEQ ID NO: 175).For detection of Axin-2, following two primers were used: Axin2 F (MUS):CAGGAGCCTCACCCTTCG (SEQ ID NO: 138), and Axin2 R (MUS):ACGCCGAGGTGCTTGCCC (SEQ ID NO: 139). Mouse GAPDH primers: mGAPDH5:CACCATGGAGAAGGCCGGGGCCCAC (SEQ ID NO: 140), and mGAPDH3:ATCATACTTGGCAGGTTTCTCCAGG (SEQ ID NO: 141) were used for the detectionof housekeeping gene expression. The reaction mixture of PCR wasprepared by adding sterilized distilled water to 2.5 ul of 10×LA PCRBuffer II (Takara Shuzo), 4 ul of 2.5 mM each dNTP Mixture (TakaraShuzo), 0.5 ul of 10 mM each primer, 2 ul of 10× cDNA diluted withsterilized distilled water and 0.5 ul of LA Taq (Takara Shuzo) to make25 ul. For detection of GIPF variant and Axin-2, reaction mixture of PCRwas incubated at 94° C. for 2 mins, reaction of 33 cycles was carriedout, with 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 30seconds. For detection of GAPDH, reaction mixture of PCR was incubatedat 94° C. for 2 mins, reaction of 23 cycles was carried out, with 94° C.for 30 seconds, 65° C. for 30 seconds and 72° C. for 30 seconds.

GIPF variant gene expression was detected in ileum and colon fromPSVR-KI mice. The expression level of Axin-2 was increased in PSVR-KImouse compared to control (FIG. 79). This data suggested that GIPFvariant had activity to induce β-catenin targeted gene expression andresponsible for activation of down stream of β-catenin signalingpathway.

Thus it indicated that GIPF variant had activity to stimulate β-cateninsignaling pathway.

Example 37 CD4+CD45RB^(high) T-Cell Transfer Model

Induction of chronic intestinal inflammation similar to inflammatorybowel disease by transferring CD4+CD45RB^(high) T lymphocyte subsets toimmunodeficient SCID mice was reported by Aranda et al., (J Immunol158:3464-3473). Accordingly, the therapeutic efficacy of GIPF toimmunologically induced colitis was tested in a CD4+CD45RB^(high) T-celltransferred model as follows.

Adult female C.B-17/Icr Crj-scid/scid mice (CHARLES RIVER JAPAN, INC.),aged 7 weeks were used. On delivery from the supplier and prior toexperiment, the animals were housed for two weeks in individuallyventilated cages on a 12 hour light:dark cycle to atbilize the circadianrhythm. Animals were allowed to food and water ad libitum.

At the day cell transfer (day 0), CD4+CD45RB^(high) T-cells werecollected form spleens of 20 BALB/c female mice. Splenocytes weresuspended in 2% FBS, 2 mM EDTA, PBS sorting buffer and CD4 positivecells were sorted by FACS (Becton Dickinson FACS Aria). Then sorted CD4positive cells were resuspended in sorting buffer to 2×10⁷ cells/ml andCD4 positive and CD45RB^(high) cell fraction was gated. CollectedCD4+CD45RB^(high) T-cells were centrifuged and suspended in 6 ml of PBSto 1.1×10⁶ cells/ml. 4.5×10⁵ cells/mouse of CD4+CD45RB^(high) T-cellswere transferred to 13 female C.B-17/Icr Crj-scid/scid mice by ipinjection.

-   -   After T-cell transfer, serum amyloid A(SAA) concentration was        monitored for inflammation progress. Also, progression of        colitis was monitored by reduction of body weight, stool        consistency and occult blood test. SAA concentration was        increased from 7 days after transfer and inflammatory was        elucidated in T-cell transferred mice. 12 days after T-cell        transfer, loose stool and occult blood positive individuals were        appeared. 30 days after T-cell transfer, diarrhea and gross        bleeding were observed in T-cell transferred mice. Further 40        days, body weight loss was observed in T-cell transferred mice.        From day 61 after T-cell transfer, 100 ug/dose GIPF was injected        iv for 3 days to 4 of T-cell transferred mice. Also PBS was        injected iv for 3 days to 3 of T-cell transferred mice as        negative control. At 70 days after T-cell transfer, these        animals were sacrificed. The intestine was removed and fixed in        formalin. Paraffin embedded sections were stained with        hematoxylin and eosin (H&E) for histological evaluation.    -   As shown FIG. 80, H&E staining of sections from the large        intestine that injected 100 ug/dose of GIPF showed clear gland        structures, increased in the number of mitotic cell and less        necrotic cell debris compared to PBS injected mice. The        histopathological changes in large intestinal mucosa caused by        T-cell trfansfer were actually reduced by GIPF injection.        Therefore, GIPF may be used for treating patients suffering from        mucositis caused by inflammatory bowel disease including Crohn's        disease.

1. A method of stimulating epithelial cell proliferation in a mammaliansubject in need thereof comprising administering to the subject atherapeutically effective amount of a composition comprising apolypeptide with epithelial cell proliferation stimulating activity,wherein said polypeptide comprises (i) an amino acid sequence with atleast 90% sequence identity to the amino acid sequence of SEQ ID NO:4,or (ii) a fragment thereof.
 2. The method of claim 1, wherein thepolypeptide comprises an amino acid sequence with at least 90% sequenceidentity to SEQ ID NO:10.
 3. The method of claim 2, wherein thepolypeptide consists of the amino acid sequence of SEQ ID NO:10.
 4. Themethod of claim 1, wherein the method comprises stimulating epithelialcell proliferation in the oral or gastrointestinal tract.
 5. The methodof claim 4, wherein the method comprises stimulating epithelial cellproliferation in the esophagus.
 6. The method of claim 4, wherein themethod comprises stimulating epithelial cell proliferation in the smallintestine.
 7. The method of claim 4, wherein the method comprisesstimulating epithelial cell proliferation in the large intestine.
 8. Themethod of claim 4, wherein the method comprises stimulating epithelialcell proliferation in the stomach.
 9. The method of claim 4, wherein themethod comprises stimulating epithelial cell proliferation in the oralcavity.
 10. The method of claim 4, wherein the subject has undergone orwill undergo radiation therapy or chemotherapy.
 11. The method of claim1, wherein the mammalian subject is a human.
 12. A method of treating agastrointestinal disorder or oral mucosal disorder in a mammaliansubject in need thereof comprising administering to the subject atherapeutically effective amount of a composition comprising apolypeptide with epithelial cell proliferation stimulating activity,wherein said polypeptide comprises (i) an amino acid sequence with atleast 90% sequence identity to the amino acid sequence of SEQ ID NO:4,or (ii) a fragment thereof.
 13. The method of claim 12, wherein thepolypeptide comprises an amino acid sequence with at least 90% sequenceidentity to SEQ ID NO:10.
 14. The method of claim 13, wherein thepolypeptide consists of the amino acid sequence of SEQ ID NO:10.
 15. Themethod of claim 12, wherein the disorder is mucositis, inflammatorybowel disease, or short bowel syndrome.